Recombinant protective protein from streptococcus pneumoniale

ABSTRACT

The present invention discloses amino acid sequences and nucleic acid sequences relating to a  Streptococcus Pneumoniae  surface associated Pneumo Protective Protein (PPP) having a molecular weight of about 20 kilo Daltons (kDa). The PPP exhibits the ability to reduce colonization of pneumococcal bacteria. Thus the present invention also pertains to compositions for the treatment and prophylaxis of infection or inflammation associated with bacterial infection.

PRIORITY

[0001] This application claims priority under 35 U.S.C. § 119 from U.S.Provisional Patent Application Serial No. 60/258,841, filed Dec. 28,2000; which is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

[0002] The present invention provides amino acid sequences and nucleicacid sequences relating to a protein of Streptococcus pneumoniae havinga molecular weight of 20 kilo Daltons (kDa). The present invention alsopertains to compositions for the treatment and prophylaxis of infectionor inflammation associated with bacterial infection.

BACKGROUND OF THE INVENTION

[0003] The middle ear is a sterile, air-filled cavity separated from theouter ear by the eardrum. Attached to the eardrum are three ear bonesthat vibrate when sound waves strike the eardrum. Vibrations aretransmitted to the inner ear, which generates nerve impulses that aresent to the brain. Air may enter the middle ear through the Eustachiantube, which opens in the walls of the nasopharynx.

[0004] The nasopharynx is located posterior to the nasal cavities. Thenasopharynx is lined by the respiratory epithelium and stratifiedsquamous epithelium. Beneath the respiratory epithelium, the abundantmucosa-associated lymphoid tissue (MALT) forms the nasopharyngeal tonsil(adenoids).

[0005] Bacterial infection or inflammation of the middle ear is mainlyobserved in children. Due to the isolation of the middle ear, it issuggested that development of middle ear infections requires theinvolvement of the nasopharynx and Eustachian tube. Infections withStreptococcus pneumoniae (S. pneumoniae) are one of the major causes ofmiddle ear infections, as well as bacteremia, meningitis, and fatalpneumonia worldwide (Butler, J. C., et al., American Journal ofMedicine, 1999, 107:69S-76S). The rapid emergence of multi-drugresistant pneumococcal strains throughout the world has led to increasedemphasis on prevention of pneumococcal infections by vaccination(Goldstein and Garau, Lancet, 1997, 350:233-4).

[0006] Protein antigens of S. pneumoniae have been evaluated forprotective efficacy in animal models of pneumococcal infection. Some ofthe most commonly studied vaccine candidates include the the PspAproteins, PsaA lipoprotein, and the CbpA protein. Numerous studies haveshown that PspA protein is a virulence factor (Crain, M. J., et al.,Infect Immun, 1990, 58:3293-9; McDaniel, L. S., et al., J Exp Med,1984,160:386-97), but is antigenically variable among pneumococcal strains.Additionally, a recent study has indicated that some antigenicallyconserved regions of a recombinant PspA variant may elicitcross-reactive antibodies in human adults (Nabors, G. S., et al.,Vaccine, 2000, 18:1743-1754). PsaA, a 37 kDa lipoprotein with similarityto other Gram-positive adhesins, is involved in manganese transport inpneumococci (Dintilhac, A., et al., Molecular Microbiology, 1997,25(4):727-739; Sampson, J. S., et al., Infect Immun, 1994, 62:319-24.)and has been shown to be protective in mouse models of systemic disease(Talkington, D. F., et al., Microb Pathog, 1996. 21:17-22). The surfaceexposed choline binding protein, CbpA, is antigenically conserved andalso is protective in mouse models of pneurnococcal disease (Rosenow,C., et al. Molecular Microbiology, 1997, 25:819-29). Sincenasopharyngeal colonization is a prerequisite for otic disease,intranasal immunization of mice with pneumococcal proteins andappropriate mucosal adjuvants has been used to enhance the mucosalantibody response and thus, the effectiveness of protein vaccinecandidates (Briles, D. E., et al., Infect Immun, 2000, 68:796-800;Yamamoto, M., et al., A. J Immunol, 1998, 161:4115-21).

[0007] The currently available 23-valent pneumococcal capsularpolysaccharide vaccine is not effective in children of less than 2 yearsof age or in immunocompromised patients, two of the major populations atrisk from pneumococcal infection (Douglas, R. M., et al., Journal ofInfectious Diseases, 1983, 148:131-137). A 7-valent pneumococcalpolysaccharide-protein conjugate vaccine, was shown to be highlyeffective in infants and children against systemic pneumococcal diseasecaused by the vaccine serotypes and against cross-reactive capsularserotypes (Shinefield and Black, Pediatr Infect Dis J, 2000, 19:394-7).The seven capsular types cover greater than 80% of the disease isolatesin the United States, but only 57-60% of disease isolates in other areasof the world (Hausdorff, W. P., et al., Clinical Infectious Diseases,2000, 30:100-21). Therefore, there is an immediate need for a vaccine tocover most or all of the disease causing serotypes of pneumococci.

[0008] Iron is an essential element for colonization and infection bymany pathogenic bacteria. Prevention of the acquisition process shouldresult in a reduction of colonization and a lower disease potential.Iron acquisition complexes in successful pathogens such as, but notlimited to, N. gonorrheae, N. meningitidis, M. catarrhalis, and H.influenzae have been evaluated for their vaccine potential by otherlaboratories (Conte, M. P, et al., Infection and Immunity, 1999,64:3925; Gray-Owens, S. D., et al. Infection and Immunity, 1995, 64:1201; Luke N. R. et al., Infection and Immunity, 1999, 67:681; Pettersson,A, et al., Infection and Immunity, 1993, 61: 4724). Thus, isolation ofthe structures responsible for iron acquisition could lead to vaccinecandidates.

SUMMARY OF THE INVENTION

[0009] The present invention contemplates an isolated S. pneumoniaesurface associated Pneumo Protective Protein (PPP) having a molecularweight of about 20 kilo Daltons (kDa), where the molecular weight isdetermined using a 10-20% SDS-PAGE gel, or a fragment thereof; the PPPhaving the ability to reduce colonization of pneumococcal bacteria.

[0010] The present invention contemplates a recombinant S. pneumoniaesurface associated PPP having a molecular weight of about 20 kDa, wherethe molecular weight is determined using a 10-20% SDS-PAGE gel, or afragment thereof; the PPP having the ability to reduce colonization ofpneumococcal bacteria.

[0011] The present invention contemplates a recombinant S. pneumoniaesurface associated PPP having a molecular weight of about 20 kDa, wherethe molecular weight is determined using a 10-20% SDS-PAGE gel, or afragment thereof; the PPP having the ability to reduce colonization ofpneumococcal bacteria; where the PPP has an isoelectric point of about4.587.

[0012] The present invention contemplates a recombinant S. pneumoniaesurface associated PPP having a molecular weight of about 20 kDa, wherethe molecular weight is determined using a 10-20% SDS-PAGE gel, or afragment thereof; the PPP having the ability to reduce colonization ofpneumococcal bacteria; where the PPP has an isoelectric point of about4.587 and a charge of about −14.214 at pH 7.

[0013] The present invention also contemplates an isolated S. pneumoniaesurface associated PPP having a molecular weight of about 20 kDa, wherethe molecular weight is determined using a 10-20% SDS-PAGE gel, or afragment thereof; where the PPP has an amino acid sequence as depictedin SEQ ID NO: 5, or a fragment thereof; the PPP having the ability toreduce colonization of pneumococcal bacteria.

[0014] The present invention also contemplates a nucleic acid sequenceencoding an isolated S. pneumoniae surface associated PPP having amolecular weight of about 20 kDa, where the molecular weight isdetermined using a 10-20% SDS-PAGE gel, or a fragment thereof; where thenucleic acid sequence has a sequence as depicted in SEQ ID NO: 4, or afragment thereof; the PPP having the ability to reduce colonization ofpneumococcal bacteria.

[0015] The present invention also contemplates a cDNA encoding anisolated S. pneumoniae surface associated PPP having a molecular weightof about 20 kDa, where the molecular weight is determined using a 10-20%SDS-PAGE gel, or a fragment thereof; where the nucleic acid sequence hasa sequence as depicted in SEQ ID NO: 4, or a fragment thereof; the PPPhaving the ability to reduce colonization of pneumococcal bacteria.

[0016] The present invention contemplates an expression vectorcomprising a nucleic acid sequence encoding an isolated S. pneumoniaesurface associated PPP having a molecular weight of about 20 kDa, wherethe molecular weight is determined using a 10-20% SDS-PAGE gel, or afragment thereof; the PPP having the ability to reduce colonization ofpneumococcal bacteria, where the sequence is operatively associated withan expression control sequence.

[0017] The present invention also contemplates a vector comprising anucleic acid sequence encoding an isolated S. pneumoniae surfaceassociated PPP having a molecular weight of about 20 kDa, where themolecular weight is determined using a 10-20% SDS-PAGE gel, or afragment thereof; the PPP having the ability to reduce colonization ofpneumococcal bacteria, where the sequence is operatively associated withan expression control sequence, and where the PPP has an isoelectricpoint of about 4.587.

[0018] The present invention further contemplates a vector comprising anucleic acid sequence encoding an isolated S. pneumoniae surfaceassociated PPP having a molecular weight of about 20 kDa, where themolecular weight is determined using a 10-20% SDS-PAGE gel, or afragment thereof; the PPP having the ability to reduce colonization ofpneumococcal bacteria, where the sequence is operatively associated withan expression control sequence, and where the PPP has an isoelectricpoint of about 4.587 and a charge of about −14.214 at pH 7.

[0019] The present invention also contemplates an expression vectorcomprising a nucleic acid sequence encoding a an isolated S. pneumoniaesurface associated PPP having a molecular weight of about 20 kDa, wherethe molecular weight is determined using a 10-20% SDS-PAGE gel, or afragment thereof; where the PPP has an amino acid sequence as depictedin SEQ ID NO: 5, or a fragment thereof; and where the nucleic acidsequence is operatively associated with an expression control sequence.

[0020] The present invention also contemplates an expression vectorcomprising a nucleic acid sequence encoding a an isolated S. pneumoniaesurface associated PPP having a molecular weight of about 20 kDa, wherethe molecular weight is determined using a 10-20% SDS-PAGE gel, or afragment thereof; where the PPP has an amino acid sequence as depictedin SEQ ID NO: 5, or a fragment thereof; where the amino acid sequence isencoded by the nucleic acid sequence as depicted in SEQ ID NO: 4, or afragment thereof; and where the nucleic acid sequence is operativelyassociated with an expression control sequence.

[0021] The present invention contemplates a host cell transfected withan expression vector comprising a nucleic acid sequence encoding anisolated S. pneumoniae surface associated PPP having a molecular weightof about 20 kDa, where the molecular weight is determined using a 10-20%SDS-PAGE gel, or a fragment thereof; the PPP having the ability toreduce colonization of pneumococcal bacteria; where the sequence isoperatively associated with an expression control sequence.

[0022] The present invention further contemplates a host celltransfected with a vector comprising a nucleic acid sequence encoding anisolated S. pneumoniae surface associated PPP having a molecular weightof about 20 kDa, where the molecular weight is determined using a 10-20%SDS-PAGE gel, or a fragment thereof; where the PPP has an amino acidsequence as depicted in SEQ ID NO: 5, or a fragment thereof; the PPPhaving the ability to reduce colonization of pneumococcal bacteria;where the sequence is operatively associated with an expression controlsequence.

[0023] The present invention also contemplates a method for producingrecombinant PPP, which method comprises isolating the PPP produced by ahost cell transfected with an expression vector and cultured underconditions that provide for expression of the PPP by the vector, wherethe vector comprises a nucleic acid sequence encoding an isolated S.pneumoniae surface associated PPP having a molecular weight of about 20kDa, where the molecular weight is determined using a 10-20% SDS-PAGEgel, or a fragment thereof; the PPP having the ability to reducecolonization of pneumococcal bacteria; where the sequence is operativelyassociated with an expression control sequence.

[0024] The present invention also contemplates a method for producingrecombinant PPP, which method comprises isolating the PPP produced byhost cell transfected with a vector and cultured under conditions thatprovide for expression of the PPP by the vector, where the vectorcomprises a nucleic acid sequence encoding an isolated S. pneumoniaesurface associated PPP having a molecular weight of about 20 kDa, wherethe molecular weight is determined using a 10-20% SDS-PAGE gel, or afragment thereof where the PPP has an amino acid sequence as depicted inSEQ ID NO: 5, or a fragment thereof; the PPP having the ability toreduce colonization of pneumococcal bacteria, where the sequence isoperatively associated with an expression control sequence.

[0025] The present invention also contemplates a composition comprising(1) an isolated S. pneumoniae surface associated PPP having a molecularweight of about 20 kDa, where the molecular weight is determined using a10-20% SDS-PAGE gel, or a fragment thereof; the PPP having the abilityto reduce colonization of pneumococcal bacteria; and (2) apharmaceutically acceptable carrier.

[0026] The present invention also contemplates a composition comprising(1) an isolated S. pneumoniae surface associated PPP having a molecularweight of about 20 kDa, where the molecular weight is determined using a10-20% SDS-PAGE gel, or a fragment thereof; the PPP having the abilityto reduce colonization of pneumococcal bacteria, and which PPP has anamino acid sequence as depicted in SEQ ID NO: 5, or a fragment thereof;and (2) a pharmaceutically acceptable carrier.

[0027] The present invention contemplates a composition comprising (1) anucleic acid sequence encoding an isolated S. pneumoniae surfaceassociated PPP having a molecular weight of about 20 kDa, where themolecular weight is determined using a 10-20% SDS-PAGE gel, or afragment thereof; the PPP having the ability to reduce colonization ofpneumococcal bacteria, where the nucleic acid sequence has a sequence asdepicted in SEQ ID NO: 4, or a fragment thereof; and (2) apharmaceutically acceptable carrier.

[0028] The present invention contemplates a composition comprising (1)an expression vector comprising a nucleic acid sequence encoding anisolated S. pneumoniae surface associated PPP having a molecular weightof about 20 kDa, where the molecular weight is determined using a 10-20%SDS-PAGE gel, or a fragment thereof; the PPP having the ability toreduce colonization of pneumococcal bacteria, where the sequence isoperatively associated with an expression control sequence; and (2) apharmaceutically acceptable carrier.

[0029] The present invention also contemplates a composition comprising(1) an expression vector comprising a nucleic acid sequence encoding aan isolated S. pneumoniae surface associated PPP having a molecularweight of about 20 kDa, where the molecular weight is determined using a10-20% SDS-PAGE gel, or a fragment thereof; where the PPP has an aminoacid sequence as depicted in SEQ ID NO: 5, or a fragment thereof, andwhere the nucleic acid sequence is operatively associated with anexpression control sequence; and (2) a pharmaceutically acceptablecarrier.

[0030] The present invention also contemplates a composition comprising(1) a host cell transfected with an expression vector comprising anucleic acid sequence encoding an isolated S. pneumoniae surfaceassociated PPP having a molecular weight of about 20 kDa, where themolecular weight is determined using a 10-20% SDS-PAGE gel, or afragment thereof; the PPP having the ability to reduce colonization ofpneumococcal bacteria, where the sequence is operatively associated withan expression control sequence; and (2) a pharmaceutically acceptablecarrier.

[0031] The present invention contemplates a composition comprising (1) ahost cell transfected with a vector comprising a nucleic acid sequenceencoding an isolated S. pneumoniae surface associated PPP having amolecular weight of about 20 kDa, where the molecular weight isdetermined using a 10-20% SDS-PAGE gel, or a fragment thereof; the PPPhaving the ability to reduce colonization of pneumococcal bacteria,where the sequence is operatively associated with an expression controlsequence; where the PPP has an amino acid sequence as depicted in SEQ IDNO: 5, or a fragment thereof; and a (2) pharmaceutically acceptablecarrier.

[0032] The present invention also contemplates an immunogeniccomposition comprising (i) a S. pneumoniae surface associated PPP havinga molecular weight of about 20 kDa, where the molecular weight isdetermined using a 10-20% SDS-PAGE gel, or a fragment thereof; (ii) apharmaceutically acceptable carrier; and (iii) optionally at least oneadjuvant.

[0033] The present invention also contemplates an immunogeniccomposition comprising (i) a S. pneumoniae surface associated PPP havinga molecular weight of about 20 kDa, where the molecular weight isdetermined using a 10-20% SDS-PAGE gel, or a fragment thereof, the PPPhaving an isoelectric point of about 4.587; (ii) a pharmaceuticallyacceptable carrier; and (iii) optionally at least one adjuvant.

[0034] The present invention also contemplates an immunogeniccomposition comprising (i) a S. pneumoniae surface associated PPP havinga molecular weight of about 20 kDa, where the molecular weight isdetermined using a 10-20% SDS-PAGE gel, or a fragment thereof, PPPhaving having an isoelectric point of about 4.587 and a charge of about−14.214 at pH 7; (ii) a pharmaceutically acceptable carrier; and (iii)optionally at least one adjuvant.

[0035] The present invention also contemplates an immunogeniccomposition comprising (i) a S. pneumoniae surface associated PPP havinga molecular weight of about 20 kDa, where the molecular weight isdetermined using a 10-20% SDS-PAGE gel, or a fragment thereof, which PPPhas an amino acid sequence as depicted in SEQ ID NO: 5, or animmunogenic fragment thereof; (ii) a pharmaceutically acceptablecarrier; and (iii) optionally at least one adjuvant.

[0036] The present invention also contemplates an immunogeniccomposition comprising (i) a S. pneumoniae surface associated PPP havinga molecular weight of about 20 kDa, where the molecular weight isdetermined using a 10-20% SDS-PAGE gel, or a fragment thereof, the PPPencoded by a nucleic acid sequence having a sequence as depicted in SEQID NO: 4, or an immunogenic fragment thereof; (ii) a pharmaceuticallyacceptable carrier; and (iii) optionally at least one adjuvant.

[0037] The present invention also contemplates an immunogeniccomposition comprising (i) a S. pneumoniae surface associated PPP havinga molecular weight of about 20 kDa, where the molecular weight isdetermined using a 10-20% SDS-PAGE gel, or a fragment thereof; (ii) apharmaceutically acceptable carrier; and (iii) optionally at least oneadjuvant; where the composition elicits protective immunity from adisease caused by Streptococcus pneumoniae.

[0038] The present invention also contemplates an immunogeniccomposition comprising (i) a S. pneumoniae surface associated PPP havinga molecular weight of about 20 kDa, where the molecular weight isdetermined using a 10-20% SDS-PAGE gel, or a fragment thereof; (ii) apharmaceutically acceptable carrier; and (iii) optionally at least oneadjuvant; where the composition elicits protective immunity from adisease caused by Streptococcus pneumoniae; where the disease isselected from the group consisting of otitis media, rhinosinusitis,bacteremia, meningitis, pneumonia, and lower respiratory tractinfection.

[0039] The present invention also contemplates an immunogeniccomposition comprising (i) a S. pneumoniae surface associated PPP havinga molecular weight of about 20 kDa, where the molecular weight isdetermined using a 10-20% SDS-PAGE gel, or a fragment thereof; (ii) apharmaceutically acceptable carrier; and (iii) optionally at least oneadjuvant; where the composition elicits protective immunity from adisease caused by Streptococcus pneumoniae; where the PPP comprises anamino acid sequence as depicted in SEQ ID NO: 5, or an immunogenicfragment thereof.

[0040] The present invention also contemplates an immunogeniccomposition comprising (i) a S. pneumoniae surface associated PPP havinga molecular weight of about 20 kDa, where the molecular weight isdetermined using a 10-20% SDS-PAGE gel, or a fragment thereof where thePPP is encoded by a nucleic acid sequence as depicted in SEQ ID NO: 4,or an immunogenic fragment thereof; (ii) a pharmaceutically acceptablecarrier; and (iii) optionally at least one adjuvant; where thecomposition elicits protective immunity from a disease caused byStreptococcus pneumoniae.

[0041] The present invention contemplates an immunogenic compositioncomprising (i) at least one expression vector encoding a PPP having amolecular weight of about 20 kDa, where the molecular weight isdetermined using a 10-20% SDS-PAGE gel; (ii) a pharmaceuticallyacceptable carrier; and (iii) optionally at least one adjuvant.

[0042] The present invention contemplates an immunogenic compositioncomprising (i) at least one expression vector encoding a PPP having amolecular weight of about 20 kDa, where the molecular weight isdetermined using a 10-20% SDS-PAGE gel; (ii) a pharmaceuticallyacceptable carrier; and (iii) optionally at least one adjuvant; wherethe pneumococcal bacteria is Streptococcus pneumoniae.

[0043] The present invention contemplates an immunogenic compositioncomprising (i) at least one expression vector encoding a PPP having amolecular weight of about 20 kDa, where the molecular weight isdetermined using a 10-20% SDS-PAGE gel; (ii) a pharmaceuticallyacceptable carrier; and (iii) optionally at least one adjuvant; wherethe composition elicits protective immunity from a disease caused byStreptococcus pneumoniae.

[0044] The present invention contemplates an immunogenic compositioncomprising (i) at least one expression vector encoding a PPP having amolecular weight of about 20 kDa, where the molecular weight isdetermined using a 10-20% SDS-PAGE gel; (ii) a pharmaceuticallyacceptable carrier; and (iii) optionally at least one adjuvant; wherethe composition elicits protective immunity from a disease caused byStreptococcus pneumoniae; where the disease is selected from the groupconsisting of otitis media, rhinosinusitis, bacterenia, meningitis,pneumonia, and lower respiratory tract infection.

[0045] The present invention contemplates an immunogenic compositioncomprising (i) at least one expression vector encoding a PPP having amolecular weight of about 20 kDa, where the molecular weight isdetermined using a 10-20% SDS-PAGE gel, where the PPP has an isoelectricpoint of about 4.587; (ii) a pharmaceutically acceptable carrier; and(iii) optionally at least one adjuvant.

[0046] The present invention contemplates an immunogenic compositioncomprising (i) at least one expression vector encoding a PPP having amolecular weight of about 20 kDa, where the molecular weight isdetermined using a 10-20% SDS-PAGE gel, where the PPP has an isoelectricpoint of about 4.587 and has a charge of about 14.214 at pH7; (ii) apharmaceutically acceptable carrier; and (iii) optionally at least oneadjuvant.

[0047] The present invention contemplates an immunogenic compositioncomprising (i) at least one expression vector encoding a PPP having amolecular weight of about 20 kDa, where the molecular weight isdetermined using a 10-20% SDS-PAGE gel where expression vector comprisesa nucleic acid sequence encoding an amino acid sequence as depicted inSEQ ID NO: 5, or an immunogenic fragment thereof; (ii) apharmaceutically acceptable carrier; and (iii) optionally at least oneadjuvant.

[0048] The present invention contemplates an immunogenic compositioncomprising (i) at least one expression vector encoding a PPP having amolecular weight of about 20 kDa, where the molecular weight isdetermined using a 10-20% SDS-PAGE gel where the expression vectorcomprises a nucleic acid sequence encoding an amino acid sequence asdepicted in SEQ ID NO: 5, or an immunogenic fragment thereof; (ii) apharmaceutically acceptable carrier; and (iii) optionally at least oneadjuvant.

[0049] The present invention contemplates an immunogenic compositioncomprising (i) at least one expression vector encoding a PPP having amolecular weight of about 20 kDa, where the molecular weight isdetermined using a 10-20% SDS-PAGE gel where the expression vectorcomprises a nucleic acid sequence depicted in SEQ ID NO:4, or animmunogenic fragment thereof; (ii) a pharmaceutically acceptablecarrier; and (iii) optionally at least one adjuvant.

[0050] The present invention contemplates a method of inducing an immuneresponse in a mammal, the method comprising administering to the mammalan amount of a composition effective to induce an immune response in themammal; where the composition comprises (i) a S. pneumoniae surfaceassociated PPP having a molecular weight of about 20 kilo Daltons (kDa),wherein said molecular weight is determined using a 10-20% SDS-PAGE gel,or a fragment thereof; (ii) a pharmaceutically acceptable carrier; and(iii) optionally at least one adjuvant.

[0051] The present invention contemplates a method of inducing an immuneresponse in a mammal, the method comprising administering to the mammalan amount of an immunogenic composition effective to induce an immuneresponse in the mammal; where the composition comprises (i) a S.pneumoniae surface associated PPP having a molecular weight of about 20kDa, where the molecular weight is determined using a 10-20% SDS-PAGEgel, or a fragment thereof, which PPP has an amino acid sequence asdepicted in SEQ ID NO: 5, or an immunogenic fragment thereof; (ii) apharmaceutically acceptable carrier; and (iii) optionally at least oneadjuvant.

[0052] The present invention contemplates a method of inducing an immuneresponse in a mammal, the method comprising administering to the mammalan amount of an immunogenic composition effective to induce an immuneresponse in the mammal; where the composition comprises (i) at least oneexpression vector encoding a PPP having a molecular weight of about 20kDa, wherein said molecular weight is determined using a 10-20% SDS-PAGEgel, where the PPP having an isoelectric point of about 4.582; (ii) apharmaceutically acceptable carrier; and (iii) optionally at least oneadjuvant.

[0053] The present invention contemplates a method of inducing an immuneresponse in a mammal, the method comprising administering to the mammalan amount of a composition effective to induce an immune response in themammal; where the composition comprises (i) at least one expressionvector encoding a PPP having a molecular weight of about 20 kDa, whereinsaid molecular weight is determined using a 10-20% SDS-PAGE gel; (ii) apharmaceutically acceptable carrier; and (iii) optionally at least oneadjuvant; wherein said expression vector comprises a nucleic acidsequence encoding an amino acid sequence as depicted in SEQ ID NO: 5, oran immunogenic fragment thereof.

[0054] The present invention contemplates a method of inducing an immuneresponse in a mammal which is infected with pneumococcal bacteria, themethod comprising administering to the mammal an amount of a compoundeffective to inhibit binding of an amino acid sequence as depicted inSEQ ID NO: 5 to induce the immune response in the mammal.

[0055] The present invention also contemplates a method for screeningfor a compound which induces an immune response in a mammal infectedwith pneumococcal bacteria, the method comprising comparing a firstamount of binding of an amino acid sequence as depicted in SEQ ID NO: 5in the presence of the compound to a second amount of binding of anamino acid sequence as depicted in SEQ ID NO: 5 not in the presence ofthe compound; whereby a lower first amount of binding than the secondamount binding indicates that the compound may induce the immuneresponse in the mammal.

[0056] The present invention also contemplates a method for diagnosingpneumococcal bacterial infection, the method comprising comparing thelevel of PPP as depicted in SEQ ID NO: 5, or fragments thereof, insuspect sample to the level of PPP as depicted in SEQ ID NO: 5, orfragments thereof, in a control sample, whereby a higher level of thePneumo Protective Protein the suspect sample than the level of thePneumo Protective Protein in the control sample indicates that thesuspect sample comprises pneumococcal bacterial infection.

[0057] The present invention further contemplates an antibody whichbinds to Streptococcus pneumoniae PPP.

[0058] The present invention also contemplates an antibody which bindsto Streptococcus pneumoniae PPP, which selectively recognizes an aminoacid sequence as depicted in SEQ ID NO: 5, or fragments thereof.

[0059] The present invention also contemplates a chimeric antibody whichbinds to Streptococcus pneumoniae PPP.

[0060] The present invention also contemplates a humanized antibodywhich binds to Streptococcus pneumoniae PPP.

[0061] The present invention also contemplates an anti-idiotypicantibody which binds to Streptococcus pneumoniae PPP.

[0062] The present invention also contemplates an antibody which bindsto Streptococcus pneumoniae PPP, where the antibody is conjugated to apharmaceutically active compound.

[0063] The present invention also contemplates a monoclonal antibodywhich binds to Streptococcus pneumoniae PPP.

[0064] The present invention also contemplates a monoclonal antibodywhich binds to Streptococcus pneumoniae PPP, where the antibody ishumanized.

[0065] The present invention also contemplates a monoclonal antibodywhich binds to Streptococcus pneumoniae PPP, where the antibody isanti-idiotypic.

[0066] The present invention also contemplates a monoclonal antibodywhich binds to Streptococcus pneumoniae PPP, where the antibody isconjugated to a pharmaceutically active compound.

[0067] The present invention contemplates a method for inducing animmune response in a mammal, the method comprising administering to themammal an amount of an anti-idiotypic antibody which binds toStreptococcus pneumoniae PPP which is effective to induce an immuneresponse in the mammal.

[0068] The present invention contemplates a method for inducing animmune response in a mammal, the method comprising administering to themammal an amount of a monoclonal antibody which binds to Streptococcuspneumoniae PPP, where the antibody is anti-idiotypic; effective toinduce an immune response in the mammal.

[0069] The present invention contemplates a method for inducing animmune response in a mammal infected with pneumococcal bacteria, themethod comprising administering to the mammal an amount of an antibodywhich binds to Streptococcus pneumoniae PPP, where the antibody isconjugated to a pharmaceutically active compound; effective to induce animmune response in the mammal.

[0070] The present invention also contemplates a method for inducing animmune response in a mammal infected with pneumococcal bacteria, themethod comprising administering to the mammal an amount of a monoclonalantibody which binds to Streptococcus pneumoniae PPP, where the antibodyis conjugated to a pharmaceutically active compound; effective to inducean immune response in the mammal.

BRIEF DESCRIPTION OF THE DRAWINGS

[0071]FIG. 1. SDS-PAGE gel of DEAE fractions from PBS washes of S.pneumoniae strain 49136. Lane 1 is unstained standards; lane 2 isfraction #8; lane 3 is fraction #9; lane 4 is fraction #10; lane 5 isfraction #11; lane 6 is fraction #12; lane 7 is fraction #13; lane 8 isfraction #19; lane 9 is fraction #15; and lane 10 is fraction #16. Thegel in FIG. 1 shows the distinct small molecular weight band infractions #14 and #15 (lanes 8 and 9) resolved by the gel.

[0072]FIG. 2. Gel of whole cell lysate of recombinant expression ofpLP533 showing expression of the desired product. Lane 1, Bioradprestained markers; Lane 2, uninduced cells; Lane 3, induced cells.

[0073]FIG. 3. Western blot of whole cell lysates of several serotypesshowing cross reactivity and oligomer formation. Lane 1, BioradPrecision prestained markers; lane 2, type 3; lane 3, type 4; lane 4,type 9; lane 5, type 14; lane 6, type 19F; lane 7, type 18C; lane 8,type 5; and lane 9, tupe 23F.

[0074]FIG. 4. Reduction of colonization by rPPP1. Bacteria recoveredshown as Log10 CFU/gram of tissue. One standard error of the mean isshown. *values are significantly different compared to the control byTukey-Kramer statistical test.

[0075]FIG. 5. SDS-PAGE gel shows purification of rPPP1. Lane 1, Bio RadPrecision standards; lane 2, diafiltrate; lane 3, purified rPPP1.

[0076]FIG. 6. Comparison of sequences of PPP1 from serotypes of S.pneumoniae.

[0077]FIG. 7. Gel shows amplified PPP1 from in vitro and in vivocultures.

DETAILED DESCRIPTION

[0078] The proteins and nucleic acids of this invention possessdiagnostic, prophylactic and therapeutic utility for diseases caused byStreptococcus pneumoniae infection. They can be used to design screeningsystems for compounds that interfere or disrupt interaction of proteinsassociated with S. pneumoniae with iron. The nucleic acids and proteinsalso can be used in the preparation of compositions against S.pneumoniae infection and/or other pathogens when used to express foreigngenes.

[0079] In the present invention, a recombinant 20 kDa protein from wholeS. pneumoniae that reduces colonization of S. pneumoniae, in anintranasal challenge model, has been identified. The protein describedherein has been named Pneumo Protective Protein 1 (PPP1 ). This proteinshows significant homology to a non heme containing ferretin proteinfrom L. innocua, which interestingly, is a member of the Dps family ofDNA binding proteins (Pikis, A., et al., J. Infect. Diseases, 1998,178:700). The ability of this protein to reduce colonization was thusunexpected, due to its predicted location in the cytoplasm.

[0080] Chemical studies indicate that the isolated S. pneumoniae surfaceassociated PPP has a molecular weight of about 20 kDa, where themolecular weight is determined using a 10-20% SDS-PAGE gel. Therecombinant PPP is determined to have an isoelectric point of about4.587. Additionally, the protein has a charge of about −14.214 at pH ofabout 7.

Streptococcus Pneumoniae

[0081]S. pneumoniae is a species of bacteria which is highly infectiousin the human body. There have been more than 80 serotypes identified, todate. Several of these serotypes are etiological agents in a variety ofdisease states including, but not limited to, pneumonia, meningitis,endocarditis, arthritis, sinusitis, otitis, bronchitis, and laryngitis.Pneumococcal infections have been identified as a leading cause of deathin persons with immunocompromised systems, such as those infected withHIV.

[0082]S. pneumoniae is a species of the Streptococcus genus of theStreptococceaceae family. This family comprises Gram-positive,non-motile, spherical or oval cells that do not form endospores. S.pneumoniae have an inorganic terminal electron acceptor foroxidative-metabolism; however, they will grow in the presence of oxygen.This allows S. pneumoniae to grow in a variety of environments and thusit is well adapted to grow in various human tissues. The bacteria isdifficult to target with penicillin, since many strains produce apolysaccharide capsule.

[0083] The first step towards pneumococcal infection is colonization ofthe nasopharynx. Disruption of binding of the pneumococci to humannasopharyngeal/otic cells should result in reduction of colonization anda lower disease potential. Thus, isolation of the structures responsiblefor pneumococcal binding to human cells could lead to vaccinecandidates. Pneumococci have evolved numerous mechanisms for binding tohuman nasopharyngeal cells, including the PspA, PsaA, and CbpA proteins.Additionally, pneumococci may specifically bind to human nasopharyngealmucin as a first step in colonization. Thus, identification of thepneumococcal structure(s) responsible for this interaction may identifypotential vaccine targets.

Molecular Biology

[0084] Embodiments of this invention relate to isolated polynucleotidesequences encoding the polypeptides or proteins, as well as variants ofsuch sequences. Preferably, under high stringency conditions, thesevariant sequences hybridize to polynucleotides encoding one or morepneumo protective proteins. More preferably, under high stringencyconditions, these variant sequences hybridize to polynucleotidesencoding one or more pneumo protective protein sequences, such as thepolynucleotide sequence of SEQ ID NO: 4. For the purposes of defininghigh stringency southern hybridization conditions, reference canconveniently be made to Sambrook et al. (1989) at pp. 387-389 which isherein incorporated by reference, where the washing step is consideredhigh stringency.

[0085] This invention also relates to conservative variants wherein thepolynucleotide sequence differs from a reference sequence through achange to the third nucleotide of a nucleotide triplet. Preferably theseconservative variants function as biological equivalents to the PPP1reference polynucleotide sequence. In a preferred embodiment, variantsthat function as biological equivalents are those that bind to iron.

[0086] The present invention further comprises DNA sequences which, byvirtue of the redundancy of the genetic code, are biologicallyequivalent to the sequences which encode for the PPP1, that is, theseother DNA sequences are characterized by nucleotide sequences whichdiffer from those set forth herein, but which encode a protein havingthe same amino acid sequence as that encoded by the DNA sequence in SEQID NO: 4.

[0087] This invention also comprises DNA sequences which encode aminoacid sequences which differ from those of the S. pneumonia PPP1, butwhich are biologically equivalent to those described for this protein(SEQ ID NO: 5). Such amino acid sequences may be said to be biologicallyequivalent to such PPP1 if their sequences differ only by minordeletions from, insertions into or substitutions to the PPP1 sequence,such that the tertiary configurations of the sequences are essentiallyunchanged from those of the wild-type protein.

[0088] For example, a codon for the amino acid alanine, a hydrophobicamino acid, may be substituted by a codon encoding another lesshydrophobic residue, such as glycine, or a more hydrophobic residue,such as valine, leucine, or isoleucine. Similarly, changes which resultin substitution of one negatively charged residue for another, such asaspartic acid for glutamic acid, or one positively charged residue foranother, such as lysine for arginine, as well as changes based onsimilarities of residues in their hydropathic index, can also beexpected to produce a biologically equivalent product. Nucleotidechanges which result in alteration of the N-terminal or C-terminalportions of the protein molecule would also not be expected to alter theactivity of the protein.

[0089] One can use the hydropathic index of amino acids in conferringinteractive biological function on a polypeptide, as discussed by Kyteand Doolittle (1982), wherein it was determined that certain amino acidsmay be substituted for other amino acids having similar hydropathicindices and still retain a similar biological activity. Alternatively,substitution of like amino acids may be made on the basis ofhydrophilicity, particularly where the biological function desired inthe polypeptide to be generated is intended for use in immunologicalembodiments. See, for example, U.S. Pat. No. 4,554,101 (which is herebyincorporated herein by reference), which states that the greatest localaverage hydrophilicity of a “protein,” as governed by the hydrophilicityof its adjacent amino acids, correlates with its immunogenicity.Accordingly, it is noted that substitutions can be made based on thehydrophilicity assigned to each amino acid. In using either thehydrophilicity index or hydropathic index, which assigns values to eachamino acid, it is preferred to introduce substitutions of amino acidswhere these values are±2, with±1 being particularly preferred, and thosewithin±0.5 being the most preferred substitutions.

[0090] Furthermore, changes in known variable regions are biologicallyequivalent where the tertiary configurations of the conserved regionsare essentially unchanged from those of PPP1. An alternative definitionof a biologically equivalent sequence is one that is still capable ofgenerating a cross-reactive immune response. In particular, the proteinsmay be modified by lengthening or shortening the corresponding insertionfrom the gonococcal pilin, as long as the modified protein is stillcapable of generating a desired immune response.

[0091] Each of the proposed modifications is well within the routineskill in the art, as is determination of retention of structural andbiological activity of the encoded products. Therefore, where the terms“pneumo protective protein”, or “PPP1”, or “PPP” are used in either thespecification or the claims, it will be understood to encompass all suchmodifications and variations which result in the production of abiologically equivalent protein.

[0092] Preferable characteristics of PPP1 described herein, encoded bythe nucleotide sequences of this invention, include one or more of thefollowing: (a) being a membrane protein or being a protein directlyassociated with a membrane; (b) capable of being separated as a proteinusing an SDS acrylamide gel; and (c) retaining its biological functionof interacting with iron.

[0093] Variants and fragments may be attenuated, i.e. having reduced onno iron-binding activity when compared to wild-type PPP1 of the presentinvention. Preferably, the fragments and variant amino acid sequencesand variant nucleotide sequences expressing PPP1 are biologicalequivalents, i.e. they retain substantially the same function of thewild-type PPP1. Such variant amino acid sequences are encoded bypolynucleotides sequences of this invention. Such variant amino acidsequences may have about 70% to about 80%, and preferably about 90%,overall similarity to the amino acid sequence of PPP1. In a preferredembodiment, these sequences are shown in FIG. 6 and SEQ ID NOs10-19. Thevariant nucleotide sequences may have either about 70% to about 80%, andpreferably about 90%, overall similarity to the nucleotide sequenceswhich, when transcribed, encode the amino acid sequence of PPP1 or avariant amino acid sequence of PPP1. The attenuated proteins of thepresent invention comprise at least one epitopic region of the wild-typeprotein. In alternative embodiments, the epitopic region of the proteincomprises at least 20 contiguous nucleotides or 8 contiguous aminoacids.

[0094] The invention further relates to the overall consensus sequenceof PPP1. Deduced amino acid sequences of PPP1 from different serotypesof S. pneumoniae may be compared to determine the conserved sequences.In a one embodiment, 10 different serotypes are compared. The conservedsequence may have many uses such as, but not limited to, determining theminimal requirements needed for protein binding, activity, and/orfunction. In a preferred embodiment, the consensus sequence of PPP1 isdepicted in FIG. 6 and SEQ ID NO:20.

[0095] The “isolated” sequences of the present invention arenon-naturally occurring sequences. For example, these sequences can beisolated from their normal state within the genome of the bacteria; orthe sequences may be synthetic, i.e. generated via recombinanttechniques, such as well-known recombinant expression systems, orgenerated by a machine.

[0096] The invention also provides a recombinant DNA cloning vehiclecapable of expressing a PPP1 comprising an expression control sequencehaving promoter and initiator sequences and a nucleic acid sequence ofthe present invention located 3′ to the promoter and initiatorsequences. Cloning vehicles can be any plasmid or expression vectorknown in the art, including viral vectors (see below). In a furtheraspect, there is provided a host cell containing a recombinant DNAcloning vehicle and/or a recombinant PPP1 of the present invention.Suitable expression control sequences, host cells and expression vectorsare well known in the art, and are described by way of example, inSambrook et al. (1989).

[0097] Suitable host cells may be selected based on factors which caninfluence the yield of recombinantly expressed proteins. These factorsinclude, but are not limited to, growth and induction conditions, mRNAstability, codon usage, translational efficiency and the presence oftranscriptional terminators to minimize promoter read through. Uponselection of suitable host cells, the cell may be transfected withexpression vectors comprising nucleic acid sequences of the presentinvention. The cells may be transfected using any methods known in theart (see below).

[0098] Once host cells have been transfected with expression vectors ofthe present invention, cells are cultured under conditions such thatpolypeptides are expressed. The polypeptide is then isolatedsubstantially free of contaminating host cell components by techniquesthat are well known to those skilled in the art.

[0099] Depending on the application of the desired recombinant proteins,a heterologous nucleotide sequence may encode a co-factor, cytokine(such as an interleukin), a T-helper epitope, a restriction marker,adjuvant, or a protein of a different microbial pathogen (e.g. virus,bacterium, fungus or parasite), especially proteins capable of elicitinga protective immune response. It may be desirable to select aheterologous sequence that encodes an immunogenic portion of aco-factor, cytokine (such as an interleukin), a T-helper epitope, arestriction marker, adjuvant, or a protein of a different microbialpathogen (e.g. virus, bacterium or fungus). Other types of non-PPP1moieties include, but are not limited to, those from cancer cells ortumor cells, allergens, amyloid peptide, protein or other macromolecularcomponents.

[0100] For example, in certain embodiments, the heterologous genesencode cytokines, such as interleukin-12, which are selected to improvethe prophylatic or therapeutic characteristics of the recombinantproteins.

[0101] Examples of such cancer cells or tumor cells include, but are notlimited to, prostate specific antigen, carcino-embryonic antigen, MUC-1,Her2, CA-125 and MAGE-3.

[0102] Examples of such allergens include, but are not limited to, thosedescribed in U.S. Pat. No. 5,830,877 and published International PatentApplication Number WO 99/51259, which are hereby incorporated byreference, and include pollen, insect venoms, animal dander, fungalspores and drugs (such as penicillin). Such components interfere withthe production of IgE antibodies, a known cause of allergic reactions.

[0103] Amyloid peptide protein (APP) has been implicated in diseasesreferred to variously as Alzheimer's disease, amyloidosis oramyloidogenic disease. The β-amyloid peptide (also referred to as Aβpeptide) is a 42 amino acid fragment of APP, which is generated byprocessing of APP by the β and γ secretase enzymes, and has thefollowing sequence:

[0104] Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln LysLeu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile Gly LeuMet Val Gly Gly Val Val Ile Ala (SEQ ID NO: 6).

[0105] In some patients, the amyloid deposit takes the form of anaggregated Aβ peptide. Surprisingly, it has now been found thatadministration of isolated Aβ peptide induces an immune response againstthe Aβ peptide component of an amyloid deposit in a vertebrate host (SeePublished International Patent Application WO 99/27944). Such Aβpeptides have also been linked to unrelated moieties. Thus, theheterologous nucleotides sequences of this invention include theexpression of this Aβ peptide, as well as fragments of Aβ peptide andantibodies to Aβ peptide or fragments thereof. One such fragment of Aβpeptide is the 28 amino acid peptide having the following sequence (asdisclosed in U.S. Pat. No. 4,666,829):

[0106] Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln LysLeu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys (SEQ ID NO: 7).

[0107] The heterologous nucleotide sequence can be selected to make useof the normal route of infection of pneumococcal bacteria, which entersthe body through the respiratory tract and can infect a variety oftissues and cells, for example, the meninges, blood, and lung. Theheterologous gene may also be used to provide agents which are used forgene therapy or for the targeting of specific cells. As an alternativeto merely taking advantage of the normal cells exposed during the normalroute of pneumococcal infection, the heterologous gene, or fragment, mayencode another protein or amino acid sequence from a different pathogenwhich, when employed as part of the recombinant protein, directs therecombinant protein to cells or tissue which are not in the normal routeof infection. In this manner, the protein becomes a targeting tool forthe delivery of a wider variety of foreign proteins.

[0108] Molecular weight of proteins may be determined by using anymethod known in the art. A non-limiting list of methods includes,denaturing SDS-PAGE gel, size exclusion chromatography, and iso-electricfocusing. Conditions appropriate for each method (e.g. time ofseparation, voltage, current, and buffers) can be determined as neededusing defined methods in the art. In a preferred embodiment, denaturingSDS-PAGE is used to determine the molecular weight of the proteins.Additionally, the conditions used to determine the molecular weight arepreferably, 1 hour separation time at 20 milli Amps and constantcurrent.

[0109] Detection of the proteins can be determined using various methodsin the art. These methods include, but are not limited to, Westernblotting, coomassie blue staining, silver staining, autoradiography,fluorescent and phosphorescent probing. In a preferred embodiment ofthis invention, the proteins were detected by Western blotting.

[0110] The terms “pneumo protective protein”, “PPP1”, and “PPP” indescribing embodiments of the invention, infra, includes embodimentsthat employ fragments, variants and attenuated forms thereof as areplacement for wild-type PPP1 or as addition thereto, unless specifiedotherwise.

Viral and Non-Viral Vectors

[0111] Preferred vectors, particularly for cellular assays in vitro andin vivo, are viral vectors, such as lentiviruses, retroviruses, herpesviruses, adenoviruses, adeno-associated viruses, vaccinia virus,baculovirus, alphaviruses and other recombinant viruses with desirablecellular tropism. Thus, a gene encoding a functional or mutant proteinor polypeptide domain fragment thereof can be introduced in vivo, exvivo, or in vitro using a viral vector or through direct introduction ofDNA. Expression in targeted tissues can be effected by targeting thetransgenic vector to specific cells, such as with a viral vector or areceptor ligand, or by using a tissue-specific promoter, or both.Targeted gene delivery is described in PCT Publication No. WO 95/28494.

[0112] Viral vectors commonly used for in vivo or ex vivo targeting andtherapy procedures are DNA-based vectors and retroviral vectors. Methodsfor constructing and using viral vectors are known in the art (e.g.,Miller and Rosman, BioTechniques, 1992, 7:980-990). Preferably, theviral vectors are replication-defective, that is, they are unable toreplicate autonomously in the target cell. Preferably, the replicationdefective virus is a minimal virus, i.e., it retains only the sequencesof its genome which are necessary for encapsulating the genome toproduce viral particles.

[0113] Examples of alphaviruses include, but are not limited to, EasternEquine Encephalitis virus (EEE), Venezuelan Equine Encephalitis virus(VEE), Everglades virus, Mucambo virus, Pixuna virus, Western EquineEncephalitis virus (WEE), Sindbis virus, Semliki Forest virus,Middelburg virus, Chikungunya virus, O'nyong-nyong virus, Ross Rivervirus, Barmah Forest virus, Getah virus, Sagiyama virus, Bebaru virus,Mayaro virus, Una virus, Aura virus, Whataroa virus, Babanki virus,Kyzylagach virus, Highlands J virus, Fort Morgan virus, Ndumu virus, andBuggy Creek virus (U.S. Pat. No. 6,156,558).

[0114] DNA viral vectors include an attenuated or defective DNA virus,such as but not limited to herpes simplex virus (HSV), papillomavirus,Epstein Barr virus (EBV), adenovirus, adeno-associated virus (AAV), andthe like. Defective viruses, which entirely or almost entirely lackviral genes, are preferred. Defective virus is not infective afterintroduction into a cell. Use of defective viral vectors allows foradministration to cells in a specific, localized area, without concernthat the vector can infect other cells. Thus, a specific tissue can bespecifically targeted. Examples of particular vectors include, but arenot limited to, a defective herpes virus 1 (HSV1) vector (Kaplitt etal., Molec. Cell. Neurosci., 1991, 2:320-330), defective herpes virusvector lacking a glyco-protein L gene, or other defective herpes virusvectors (PCT Publication Nos. WO 94/21807 and WO 92/05263); anattenuated adenovirus vector, such as the vector described byStratford-Perricaudet et al. (J. Clin. Invest., 1992, 90:626-630; seealso La Salle et al., Science, 1993, 259:988-990); and a defectiveadeno-associated virus vector (Samulski et al., J. Virol., 1987,61:3096-3101; Samulski et al., J. Virol., 1989, 63:3822-3828; Lebkowskiet al., Mol. Cell. Biol., 1988, 8:3988-3996).

[0115] Various companies produce viral vectors commercially, including,but not limited to, Avigen, Inc. (Alameda, Calif.; AAV vectors), CellGenesys (Foster City, Calif.; retroviral, adenoviral, AAV vectors, andlentiviral vectors), Clontech (retroviral and baculoviral vectors),Genovo, Inc. (Sharon Hill, Pa.; adenoviral and AAV vectors), Genvec(adenoviral vectors), IntroGene (Leiden, Netherlands; adenoviralvectors), Molecular Medicine (retroviral, adenoviral, AAV, and herpesviral vectors), Norgen (adenoviral vectors), Oxford BioMedica (Oxford,United Kingdom; lentiviral vectors), and Transgene (Strasbourg, France;adenoviral, vaccinia, retroviral, and lentiviral vectors).

[0116] Adenovirus Vectors.

[0117] Adenoviruses are eukaryotic DNA viruses that can be modified toefficiently deliver a nucleic acid of the invention to a variety of celltypes. Various serotypes of adenovirus exist. Of these serotypes,preference is given, within the scope of the present invention, to usingtype 2 or type 5 human adenoviruses (Ad 2 or Ad 5) or adenoviruses ofanimal origin (see PCT Publication No. WO 94/26914). Those adenovirusesof animal origin which can be used within the scope of the presentinvention include adenoviruses of canine, bovine, murine (example: Mav1,Beard et al., Virology, 1990, 75-81), ovine, porcine, avian, and simian(example: SAV) origin. Preferably, the adenovirus of animal origin is acanine adenovirus, more preferably a CAV2 adenovirus (e.g., Manhattan orA26/61 strain, ATCC VR-800, for example). Various replication defectiveadenovirus and minimum adenovirus vectors have been described (PCTPublication Nos. WO 94/26914, WO 95/02697, WO 94/28938, WO 94/28152, WO94/12649, WO 95/02697, WO 96/22378). The replication defectiverecombinant adenoviruses according to the invention can be prepared byany technique known to the person skilled in the art (Levrero et al.,Gene, 1991, 101:195; European Publication No. EP 185 573; Graham, EMBOJ., 1984, 3:2917; Graham et al., J. Gen. Virol., 1977, 36:59).Recombinant adenoviruses are recovered and purified using standardmolecular biological techniques, which are well known to one of ordinaryskill in the art.

[0118] Adeno-Associated Viruses.

[0119] The adeno-associated viruses (AAV) are DNA viruses of relativelysmall size that can integrate, in a stable and site-specific manner,into the genome of the cells which they infect. They are able to infecta wide spectrum of cells without inducing any effects on cellulargrowth, morphology or differentiation, and they do not appear to beinvolved in human pathologies. The AAV genome has been cloned, sequencedand characterized. The use of vectors derived from the AAVs fortransferring genes in vitro and in vivo has been described (see, PCTPublication Nos. WO 91/18088 and WO 93/09239; U.S. Pat. Nos. 4,797,368and 5,139,941; European Publication No. EP 488 528). The replicationdefective recombinant AAVs according to the invention can be prepared bycotransfecting a plasmid containing the nucleic acid sequence ofinterest flanked by two AAV inverted terminal repeat (ITR) regions, anda plasmid carrying the AAV encapsidation genes (rep and cap genes), intoa cell line which is infected with a human helper virus (for example anadenovirus). The AAV recombinants which are produced are then purifiedby standard techniques.

[0120] Retrovirus Vectors.

[0121] In another embodiment the gene can be introduced in a retroviralvector, e.g., as described in U.S. Pat. No. 5,399,346; Mann et al, Cell,1983, 33:153; U.S. Pat. Nos. 4,650,764 and 4,980,289; Markowitz et al.,J. Virol., 1988, 62:1120; U.S. Pat. No. 5,124,263; European PublicationNos. EP 453 242 and EP178 220; Bernstein et al., Genet. Eng.,1985,7:235;McCormick, BioTechnology, 1985, 3:689; PCT Publication No. WO 95/07358;and Kuo et at., Blood, 1993, 82:845. The retroviruses are integratingviruses that infect dividing cells. The retrovirus genome includes twoLTRs, an encapsidation sequence and three coding regions (gag, pol andenv). In recombinant retroviral vectors, the gag, pol and env genes aregenerally deleted, in whole or in part, and replaced with a heterologousnucleic acid sequence of interest. These vectors can be constructed fromdifferent types of retrovirus, such as, HIV, MoMuLV (“murine Moloneyleukaemia virus” MSV (“murine Moloney sarcoma virus”), HaSV (“Harveysarcoma virus”); SNV (“spleen necrosis virus”); RSV (“Rous sarcomavirus”) and Friend virus. Suitable packaging cell lines have beendescribed in the prior art, in particular the cell line PA317 (U.S. Pat.No. 4,861,719); the PsiCRIP cell line (PCT Publication No. WO 90/02806)and the GP+envAm−12 cell line (PCT Publication No. WO 89/07150). Inaddition, the recombinant retroviral vectors can contain modificationswithin the LTRs for suppressing transcriptional activity as well asextensive encapsidation sequences which may include a part of the gaggene (Bender et al., J. Virol., 1987, 61:1639). Recombinant retroviralvectors are purified by standard techniques known to those havingordinary skill in the art.

[0122] Retroviral vectors can be constructed to function as infectiousparticles or to undergo a single round of transfection. In the formercase, the virus is modified to retain all of its genes except for thoseresponsible for oncogenic transformation properties, and to express theheterologous gene. Non-infectious viral vectors are manipulated todestroy the viral packaging signal, but retain the structural genesrequired to package the co-introduced virus engineered to contain theheterologous gene and the packaging signals. Thus, the viral particlesthat are produced are not capable of producing additional virus.

[0123] Retrovirus vectors can also be introduced by DNA viruses, whichpermits one cycle of retroviral replication and amplifies tranfectionefficiency (see PCT Publication Nos. WO 95/22617, WO 95/2641 1, WO96/39036 and WO 97/19182).

[0124] Lentivirus Vectors.

[0125] In another embodiment, lentiviral vectors can be used as agentsfor the direct delivery and sustained expression of a transgene inseveral tissue types, including brain, retina, muscle, liver and blood.The vectors can efficiently transduce dividing and nondividing cells inthese tissues, and maintain long-term expression of the gene ofinterest. For a review, see, Naldini, Curr. Opin. Biotechnol., 1998,9:457-63; see also Zufferey, et al., J. Virol., 1998, 72:9873-80).Lentiviral packaging cell lines are available and known generally in theart. They facilitate the production of high-titer lentivirus vectors forgene therapy. An example is a tetracycline-inducible VSV-G pseudotypedlentivirus packaging cell line that can generate virusparticles attiters greater than 106 IU/ml for at least 3 to 4 days (Kafri, et al.,J. Virol., 1999, 73: 576-584). The vector produced by the inducible cellline can be concentrated as needed for efficiently transducingnon-dividing cells in vitro and in vivo.

[0126] Non-Viral Vectors.

[0127] In another embodiment, the vector can be introduced in vivo bylipofection, as naked DNA, or with other transfection facilitatingagents (peptides, polymers, etc.). Synthetic cationic lipids can be usedto prepare liposomes for in vivo transfection of a gene encoding amarker (Felgner, et. al., Proc. Natl. Acad. Sci. U.S.A., 1987,84:7413-7417; Felgner and Ringold, Science, 1989, 337:387-388; seeMackey, et al., Proc. Natl. Acad. Sci. U.S.A., 1988, 85:8027-8031; Ulmeret al., Science, 1993, 259:1745-1748). Useful lipid compounds andcompositions for transfer of nucleic acids are described in PCT PatentPublication Nos. WO 95/18863 and WO 96/17823, and in U.S. Pat. No.5,459,127. Lipids may be chemically coupled to other molecules for thepurpose of targeting (see Mackey, et. al., supra). Targeted peptides,e.g., hormones or neurotransmitters, and proteins such as antibodies, ornon-peptide molecules could be coupled to liposomes chemically.

[0128] Other molecules are also useful for facilitating transfection ofa nucleic acid in vivo, such as a cationic oligopeptide (e.g., PCTPatent Publication No. WO 95/21931), peptides derived from DNA bindingproteins (e.g., PCT Patent Publication No. WO 96/25508), or a cationicpolymer (e.g., PCT Patent Publication No. WO 95/21931).

[0129] It is also possible to introduce the vector in vivo as a nakedDNA plasmid. Naked DNA vectors for gene therapy can be introduced intothe desired host cells by methods known in the art, e.g.,electroporation, microinjection, cell fusion, DEAE dextran, calciumphosphate precipitation, use of a gene gun, or use of a DNA vectortransporter (e.g., Wu et al., J. Biol. Chem., 1992, 267:963-967; Wu andWu, J. Biol. Chem., 1988, 263:14621-14624; Canadian Patent ApplicationNo. 2,012,311; Williams et al., Proc. Natl. Acad. Sci. USA, 1991,88:2726-2730). Receptor-mediated DNA delivery approaches can also beused (Curiel et al., Hum. Gene Ther., 1992, 3:147-154; Wu and Wu, J.Biol. Chem., 1987, 262:4429-4432). U.S. Pat. Nos. 5,580,859 and5,589,466 disclose delivery of exogenous DNA sequences, free oftransfection facilitating agents, in a mammal. Recently, a relativelylow voltage, high efficiency in vivo DNA transfer technique, termedelectrotransfer, has been described (Mir et al., C. P. Acad. Sci., 1988,321:893; PCT Publication Nos. WO 99/01157; WO 99/01158; WO 99/01175).

Assay System

[0130] Any cell assay system that allows for assessing functionalactivities of immunogenic compositions and compounds that modulatebinding of PPP1 to iron is contemplated by the present invention. In aspecific embodiment, the assay can be used to identify compounds thatinteract with PPP1 to decrease binding of PPP1, described herein, toiron. This can be evaluated by assessing the effects of a test compoundon the interaction the protein described herein. A cell assay systemthat assesses the ability of the compound to elicit opsonophagocyticantibodies against S. pneumoniae may also be utilized (Gray, B. M. 1990.Conjugate Vaccines Supplement p694-697).

[0131] Any convenient method that permits detection of the binding ofiron with PPP are contemplated by the present invention. In a preferredembodiment of the invention, protein components of S. pneumoniae can beseparated on a polyacrylamide gel and transferred to a solid support.The support then may be probed with a labeled interacting component(e.g. iron). The component may be labeled with any label known in theart including, but not limited to, radioactivity, enzyme-based, dyemolecules, or a flourescent or phosphorescent tag. In a preferredembodiment, the label is radioactive. The label may be detected by anymeans known in the art. For example, autoradiography, scintillationcounter, or ultra-violet light. In a preferred embodiment, theradiolabel is detected by autoradiography. Assays that amplify thesignals from the probe are also known, such as, for example, those thatutilize biotin and avidin, and enzyme-labeled immunoassays, such asELISA assays.

In Vitro Screening Methods

[0132] Candidate agents are added to assay systems, prepared by knownmethods in the art, and the level of binding betwen iron and PPP1 ismeasured. Various in vitro systems can be used to analyze the effects ofa compound on iron binding. Preferably, each experiment is performedmore than once, such as, for example, in triplicate at multipledifferent dilutions of compound.

[0133] The screening system of the invention permits detection ofbinding inhibitors. An inhibitor screen involves detecting interactionof iron and PPP1 when contacted with a compound that regulatesinteraction of these proteins. If a decrease in the binding of iron toPPP1 is detected, then the compound is a candidate inhibitor. If nodecrease is observed, the compound does not alter the binding of iron tothe protein of the present invention.

Immunogenic Compositions

[0134] In further embodiments of this invention PPP1 are employed inimmunogenic compositions comprising (i) at least one PPP1; (ii) at leastone pharmaceutically acceptable buffer, diluent, or carrier; and (iii)optionally at least one adjuvant. In a preferred embodiment, theimmunogenic composition is used as a vaccine. The PPP1 may berecombinantly produced or isolated from a bacterial preparation,according to methods known in the art. Preferably, these compositionshave therapeutic and prophylactic applications as immunogeniccompositions in preventing, protecting and/or ameliorating pneumococcalinfection. In such applications, an immunologically effective amount ofat least one PPP1 is employed in such amount to cause a reduction,preferably a substantial reduction, in the course a normal pneumoccocalinfection. The proteins may be attenuated. The term “attenuated” refersto a protein that maintains its immunogenic activity, while one or moreother functional characteristics are decreased or deleted. For example,the attenuated form of this protein may exhibit diminished bindingproperties, such as its ability to bind iron. Alternatively, theattenuated form may decrease the ability of S. pneumoniae to bind iron.

[0135] As used herein, the term “effective amount” refers to amount ofthe immunogen component (i.e. PPP1) described herein to stimulate animmune response, i.e., to cause the production of antibodies and/or acell-mediated response when introduced into a subject. In a preferredembodiment, the effective amount will decrease the colonization of S.pneumoniae. The term “immunogen component” refers to the ability of thiscomponent to stimulate secretory antibody and/or cell-mediated responseproduction in local regions, e.g. nasopharynx, when administeredsystemically as an immunogenic composition according to the presentinvention.

[0136] As used herein the term “adjuvant” refers to an agent, compoundor the like, which potentiates or stimulates the immune response in asubject when administered in combination with the immunogeniccomposition. Thus, the immune response, elicited by the immunogeniccomposition combination, as measured by any convention method known inthe art, will generally be greater than that provoked by the immunogeniccomposition alone.

[0137] The compositions of the invention can include an adjuvant,including, but not limited to aluminum hydroxide; aluminum phosphate;Stimulon™ QS-21 (Aquila Biopharmaceuticals, Inc., Framingham, Mass.);MPL™ (3-O-deacylated monophosphoryl lipid A; Corixa, Seattle, Wash.);RC529 (Corixa) and aminoalkyl glucosamine phosphate compounds asdescribed in PCT Published Application WO 98/50399 (RIBI ImmunochemResearch); IL-12 (Genetics Institute, Cambridge, Mass.);N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP);N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to asnor-MDP);N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphos-phoryloxy)-ethylamine(CGP 19835A, referred to a MTP-PE); granulocyte-macrophage colonystimulating factor (GM-CSF) and cholera toxin. Others which may be usedare non-toxic derivatives of cholera toxin, including its B subunit (forexample, wherein glutamic acid at amino acid position 29 is replaced byanother amino acid, preferably, a histidine in accordance with PublishedInternational Patent Application WO 00/18434), and/or conjugates orgenetically engineered fusions of non-PPP polypeptides with choleratoxin or its B subunit, procholeragenoid, fungal polysaccharides. Theadjuvant may be used in its natural form or one can use a synthetic orsemi-synthetic version of an adjuvant. Any formulation of the adjuvantmay be used depending on the desired response and admininstrationmethod. Various forms of the adjuvant may be used, e.g., a liquid,powder or emulsion.

[0138] The immunogenic composition may be administered as a single bolusdose or as a “series” of administrations over a defined period of time(e.g., one year). When given in later year, such series ofadministrations is referred to as “booster shots”. These administrationsincrease the antibody levels produced by the previous administration.The immunogenic compound may be administered until sufficient antibodylevels have been identified in the subject, so as to induce an immuneresponse upon challenge from the immunogen.

[0139] The formulation of such immunogenic compositions is well known topersons skilled in this field. Immunogenic compositions of the inventionmay comprise additional antigenic components (e.g., polypeptide orfragment thereof or nucleic acid encoding an antigen or fragmentthereof) and, preferably, include a pharmaceutically acceptable carrier.Suitable pharmaceutically acceptable carriers and/or diluents includeany and all conventional solvents, dispersion media, fillers, solidcarriers, aqueous solutions, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like. The term“pharmaceutically acceptable carrier” refers to a carrier that does notcause an allergic reaction or other untoward effect in patients to whomit is administered. Suitable pharmaceutically acceptable carriersinclude, for example, one or more of water, saline, phosphate bufferedsaline, dextrose, glycerol, ethanol and the like, as well ascombinations thereof. Pharmaceutically acceptable carriers may furthercomprise minor amounts of auxiliary substances such as wetting oremulsifying agents, preservatives or buffers, which enhance the shelflife or effectiveness of the antigen. The use of such media and agentsfor pharmaceutically active substances is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with theactive ingredient, use thereof in immunogenic compositions of thepresent invention is contemplated.

Compositions

[0140] In further embodiments of this invention, PPP1 nucleic acidsequences, amino acid sequences, expression vectors or host cells areemployed in compositions comprising (i) at least one PPP1 protein, ornucleic acid encoding an amino acid sequence of a PPP1, or an expressionvector or host cell that expresses such nucleic acid arid (ii) at leastone of a pharmaceutically acceptable buffer, diluent, or carrier. ThePPP1 may be recombinantly produced or isolated from a bacterialpreparation, according to methods known in the art. Preferably, thesecompositions have therapeutic and prophylactic applications. In suchapplications, a pharmaceutically effective amount of at least one PPP1is employed in such amount to produce a defined functional activity. Asused herein, the term “effective amount” refers to amount of the PPP1protein described herein, to produce a functional effect.

[0141] Administration of such compositons or immunogenic compositionsmay be by any conventional effective form, such as intranasally,parenterally, orally, or topically applied to mucosal surface such asintranasal, oral, eye, lung, vaginal, or rectal surface, such as byaerosol spray. The preferred means of administration is parenteral orintranasal.

[0142] Oral formulations include such normally employed excipients as,for example, pharmaceutical grades of mannitol, lactose, starch,magnesium stearate, sodium saccharine, cellulose, magnesium carbonate,and the like.

[0143] The polynucleotides and polypeptides of the present invention maybe administered as the sole active immunogen in an immunogeniccomposition. Alternatively, however, the immunogenic composition mayinclude other active immunogens, including other immunologically activeantigens from other pathogenic species. Preferably, the pathogenicspecies that provide other immunologically active antigens are bacterialpathogens, e.g., involved in bacterial infections. Indeed, preferablytherapeutic use of the PPP antigen of the invention will be as acomponent of a multivalent vaccine that includes other bacterialantigens from S. pneumonia or other pathogenic bacteria. The otherimmunologically active antigens may be replicating agents ornon-replicating agents. Replicating agents include, for example,attenuated forms of measles virus, rubella virus, variscella zostervirus (VZV), Parainfluenza virus (PIV), and Respiratory Syncytial virus(RSV).

[0144] One of the important aspects of this invention relates to amethod of inducing immune responses in a mammal comprising the step ofproviding to said mammal an immunogenic composition of this invention.The immunogenic composition is a composition which is immunogenic in thetreated animal or human such that the immunologically effective amountof the polypeptide(s) contained in such composition brings about thedesired response against pneumococcal infection. Preferred embodimentsrelate to a method for the treatment, including amelioration, orprevention of pneumococcal infection in a human comprising administeringto a human an immunologically effective amount of the immunogeniccomposition. The dosage amount can vary depending upon specificconditions of the individual. This amount can be determined in routinetrials by means known to those skilled in the art.

[0145] Certainly, the isolated amino acid sequences for the proteins ofthe present invention may be used in forming subunit immunogeniccompositions. They also may be used as antigens for raising polyclonalor monoclonal antibodies and in immunoassays for the detection ofanti-PPP1 protein-reactive antibodies. Immunoassays encompassed by thepresent invention include, but are not limited to, those described inU.S. Pat. No. 4,367,110. (double monoclonal antibody sandwich assay) andU.S. Pat. No. 4,452,901 (western blot), which U.S. Patents areincorporated herein by reference. Other assays includeimmunoprecipitation of labeled ligands and immunocytochemistry, both invitro and in vivo.

Methods of Inducing an Immune Response

[0146] According to the present invention, colonization of S. pneumoniaeinvolves PPP1 proteins. The present invention provides for methods thatprevent pneumococal infections by administering to a subject atherapeutically effective amount of an immunogenic composition thatinduces an immune response in the subject. These methods include, butare not limited to, administration of an immunogenic compositioncomprised of at least one PPP1 protein, variant, fragment or attenuatedversion thereof, or at least one expression vector encoding the proteinvariant, fragment or attenuated version thereof..

Methods of Inhibiting Pneumococcal Infection

[0147] The present invention further provides for methods to induce animmune response in a subject which is infected with pneumococal bacteriaby administering to a subject a therapeutically effective amount of acomposition or compound that blocks functional effects associated withthe PPP1 proteins. These methods include, but are not limited to,administration of a composition comprised of at least one PPP1 proteinor fragments thereof or at least one expression vector encoding a PPP1protein or administration of a compound that blocks, substantially allor at least in part, a function of the PPP1 proteins.

Methods of Diagnosis

[0148] This invention also provides for a method of diagnosing apneumococcal infection, or identifying a pneumococcal immunogeniccompositon strain that has been administered, comprising the step ofdetermining the presence, in a sample, of an amino acid sequence of SEQID NO: 5 or any of 10-19. Any conventional diagnostic method may beused. These diagnostic methods can easily be based on the presence of anamino acid sequence or polypeptide. Preferably, such a diagnostic methodmatches for a polypeptide having at least 10, and preferably at least20, amino acids which are common to the amino acid sequences of thisinvention.

[0149] The nucleic acid sequences disclosed herein also can be used fora variety of diagnostic applications. These nucleic acids sequences canbe used to prepare relatively short DNA and RNA sequences that have theability to specifically hybridize to the nucleic acid sequences encodingthe PPP1 protein. Nucleic acid probes are selected for the desiredlength in view of the selected parameters of specificity of thediagnostic assay. The probes can be used in diagnostic assays fordetecting the presence of pathogenic organisms, or in identifying apneumococcal immunogenic composition that has been administered, in agiven sample. With current advanced technologies for recombinantexpression, nucleic acid sequences can be inserted into an expressionconstruct for the purpose of screening the corresponding oligopeptidesand polypeptides for reactivity with existing antibodies or for theability to generate diagnostic or therapeutic reagents. Suitableexpression control sequences and host cell/cloning vehicle combinationsare well known in the art, and are described by way of example, inSambrook et al. (1989).

[0150] In preferred embodiments, the nucleic acid sequences employed forhybridization studies or assays include sequences that are complementaryto a nucleotide stretch of at least about 10, preferably about 15, andmore preferably about 20 nucleotides. A variety of known hybridizationtechniques and systems can be employed for practice of the hybridizationaspects of this invention, including diagnostic assays such as thosedescribed in Falkow et al., U.S. Pat. No. 4,358,535. Preferably, thesequences recognize or bind a nucleic acid sequence on the PPP1 proteinare consecutive.

[0151] In general, it is envisioned that the hybridization probesdescribed herein will be useful both as reagents in solutionhybridizations as well as in embodiments employing a solid phase. Inembodiments involving a solid phase, the test DNA (or RNA) fromsuspected clinical samples, such as exudates, body fluids (e.g., middleear effusion, bronchoalveolar lavage fluid) or even tissues, is absorbedor otherwise affixed to a selected matrix or surface. This fixed,single-stranded nucleic acid is then subjected to specific hybridizationwith selected probes under desired conditions. The selected conditionswill depend on the particular circumstances based on the particularcriteria required (depending, for example, on the G+C contents, type oftarget nucleic acid, source of nucleic acid, size of hybridizationprobe). Following washing of the hybridized surface so as to removenonspecifically bound probe molecules, specific hybridization isdetected, or even quantified, by means of the label.

[0152] The nucleic acid sequences which encode the PPP1 protein of theinvention, or their variants, may be useful in conjunction with PCR*technology, as set out, e.g., in U.S. Pat. No. 4,603,102. One mayutilize various portions of any of the PPP1 protein sequences of thisinvention as oligonucleotide probes for the PCR* amplification of adefined portion of a PPP1 gene, or nucleotide, which sequence may thenbe detected by hybridization with a hybridization probe containing acomplementary sequence. In this manner, extremely small concentrationsof the PPP1 nucleic acid sequence may be detected in a sample utilizingthe nucleotide sequences of this invention.

[0153] The following examples are included to illustrate certainembodiments of the invention. However, those of skill in the art should,in the light of the present disclosure, appreciate that many changes canbe made in the specific embodiments which are disclosed and still obtaina like or similar result without departing from the spirit and scope ofthe invention.

Antibodies

[0154] The present invention describes antibodies that may be used todetect the presence of PPP1 proteins present in samples. Additionally,the antibodies (e.g., anti-idiotypic antibodies) may be used to inhibitimmune responses to pneumococcal infections.

[0155] According to the invention, PPP1 protein polypeptides producedrecombinantly or by chemical synthesis, and fragments or otherderivatives, may be used as an immunogen to generate antibodies thatrecognize the polypeptide or portions thereof. The portion of thepolypeptide used as an immunogen may be specifically selected tomodulate immunogenicity of the developed antibody. Such antibodiesinclude, but are not limited to, polyclonal, monoclonal, humanized,chimeric, single chain, Fab fragments, and an Fab expression library. Anantibody that is specific for human PPP1 protein may recognize awild-type or mutant form of the PPP1 proteins. In a specific embodiment,the antibody is comprised of at least 8 amino acids, preferably from8-10 amino acids, and more preferably from 15-30 amino acids.Preferably, the antibody recognizes or binds amino acids on PPP1 areconsecutive.

[0156] Various procedures known in the art may be used for theproduction of polyclonal antibodies to polypeptides, derivatives, oranalogs. For the production of antibody, various host animals, includingbut not limited to rabbits, mice, rats, sheep, goats, etc, can beimmunized by injection with the polypeptide or a derivative (e.g.,fragment or fusion protein). The polypeptide or fragment thereof can beconjugated to an immunogenic carrier, e.g., bovine serum albumin (BSA)or keyhole limpet hemocyanin (KLH). Various adjuvants may be used toincrease the immunological response, depending on the host species,including but not limited to Freund's (complete and incomplete), mineralgels such as aluminum hydroxide, surface active substances such aslysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,KLH, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum.

[0157] Monoclonal antibodies directed toward a PPP1 protein, fragment,analog, or derivative thereof, may be prepared by any technique thatprovides for the production of antibody molecules by continuous celllines in culture may be used. These include but are not limited to thehybridoma technique originally developed by Kohler and Milstein (Nature256:495-497, 1975), as well as the trioma technique, the human B-cellhybridoma technique (Kozbor et al., Immunology Today 4:72, 1983; Cote etal., Proc. Natl. Acad. Sci. U.S.A. 80:2026-2030, 1983), and theEBV-hybridoma technique to produce human monoclonal antibodies (Cole etal., in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc.,pp. 77-96, 1985). “Chimeric antibodies” may be produced (Morrison etal., J. Bacteriol. 159:870, 1984; Neuberger et al., Nature 312:604-608,1984; Takeda et al., Nature 314:452-454, 1985) by splicing the genesfrom a non-human antibody molecule specific for a polypeptide togetherwith genes from a human antibody molecule of appropriate biologicalactivity.

[0158] In the production and use of antibodies, screening for or testingwith the desired antibody can be accomplished by techniques known in theart, e.g., radioimmunoassay, ELISA (enzyme-linked immunosorbant assay),“sandwich” immunoassays, immunoradiometric assays, gel diffusionprecipitin reactions, immunodiffusion assays, in situ immunoassays(using colloidal gold, enzyme or radioisotope labels, for example),western blots, precipitation reactions, agglutination assays (e.g., gelagglutination assays, hemagglutination assays), complement fixationassays, immunofluorescence assays, protein A assays, andimmunoelectrophoresis assays, etc.

[0159] The foregoing antibodies can be used in methods known in the artrelating to the localization and activity of the polypeptide, e.g., forWestern blotting, imaging the polypeptide in situ, measuring levelsthereof in appropriate physiological samples, etc. using any of thedetection techniques mentioned above or known in the art. Suchantibodies can also be used in assays for ligand binding, e.g., asdescribed in U.S. Pat. No. 5,679,582. Antibody binding generally occursmost readily under physiological conditions, e.g., pH of between about 7and 8, and physiological ionic strength. The presence of a carrierprotein in the buffer solutions stabilizes the assays. While there issome tolerance of perturbation of optimal conditions, e.g., increasingor decreasing ionic strength, temperature, or pH, or adding detergentsor chaotropic salts, such perturbations will decrease binding stability.

[0160] In a specific embodiment, antibodies that agonize the activity ofthe PPP1 protein can be generated. In particular, intracellular singlechain Fv antibodies can be used to regulate the PPP1 protein. Suchantibodies can be tested using the assays described below foridentifying ligands.

[0161] In another specific embodiment, the antibodies of the presentinvention are anti-idiotypic antibodies. These antibodies recognize andor bind to other antibodies present in the system. The anti-idiotypicantibodies may be monoclonal, polyclonal, chimeric, humanized.

[0162] In another specific embodiment, antibodies of the presentinvention are conjugated to a secondary component, such as, for example,a small molecule, polypeptide, or polynucleotide. The conjugation may beproduced through a chemical modification of the antibody, whichconjugates the antibody to the secondary component. The conjugatedantibody will allow for targeting of the secondary component, such as,for example, an antibiotic to the site of interest. The secondarycomponent may be of any size or length. In a specific embodiment, thesecondary component is a pharmaceutically active compound.

[0163] A further aspect of this invention relates to the use ofantibodies, as discussed supra, for targeting a pharmaceutical compound.In this embodiment, antibodies against the PPP1 protein are used topresent specific compounds to infected sites. The compounds, preferablyan antibiotic agent, when conjugated to the antibodies are referred toas targeted compounds or targeted agents. Methods for generating suchtarget compounds and agents are known in the art. Exemplary publicationson target compounds and their preparation are set forth in U.S. Pat.Nos. 5,053,934; 5,773,001; and 6,015,562.

EXAMPLES Materials and Methods

[0164] Bacterial Strains and Plasmids

[0165]S. pneumoniae strains utilized in this work were S. pneumoniaeCP1200, a nonencapsulated, highly transformable derivative of R36A, arough variant of D39, a virulent type 2 strain, (Morrison, D. A. et al.,J. Bacteriology, 1983, 156:281) was obtained from Margaret Hostetter atYale University, C T., and S. pneumoniae strain 49136 obtained from theATCC. S. pneumoniae were grown to log phase (approx O.D. of 0.6-0.8 at600 nm) in Todd Hewitt media (Difco Lab., Detroit, Mich.) with 0.5%yeast extract (Difco) at 37° C. with aeration or on Tryptic Soy (Difco)blood agar plates. Escherichia coli strains used in this study wereBL21(DE3), BLR(DE3) (Novagen, Madison, Wis.), Top10F′(Invitrogen, SanDiego, Calif.), and were grown in SOB media (15) at 37° C. with aerationcontaining appropriate antibiotics. Plasmids used in this work werePCR2.1 TOPO (Invitrogen) and pET28a (Novagen). Where specified,chloramphenicol was used at 20 μg/ml, ampicillin at 100 μg/ml,streptomycin at 100 μg/ml, and kanamycin at 25 μg/ml. Restrictionenzymes were purchased from New England Biolabs (Beverly, Mass.) andused according to manufactures directions.

[0166] Identification of a Surface Associated Protein in Outer MembraneFractions of S. pneumoniae

[0167] Extraction of Surface Associated Components

[0168] Bacteria were grown in 4 liters of Todd Hewitt broth, andharvested by centrifugation at 8000×g for 30 minutes. The pellet wassuspended in ˜175 ml of PBS with the aid of a pipette and immediatelycentrifuged at 20000×g for 30 min. The wash was filtered through a 0.45m filter (Nalgene, Rochester, N.Y.), dialyzed and lyophilized.

[0169] Ion-exchange Chromatography of Surface Associated ProteinComponents

[0170] The PBS extract of S. pneumoniae was dissolved in Tris-HCl, pH7.6 (10 mM, 100 ml) and subjected to ion exchange chromatography in acolumn of DEAE-Sepharose CL-6B. After washing the column with the samplebuffer, it was eluted first with 200 mM Tris-HCl, pH 7.6 followed by alinear NaCl gradient to a final NaCl concentration of 0.75 M (in 200 mMTris-HCl, pH 7.6) over 300 ml. Column fractions were analyzed bySDS-PAGE gel. Fractions containing a substantial amount of a surfaceassociated protein of approximately 18-20 kDa were pooled, desalted byCentricon SR3 concentrator and lyophilized.

[0171] N-terminal Amino Acid Sequence Analysis by PVDF Blot Excision.

[0172] The sample was diluted to 1 mg/mL total protein and combined 1:1with 2× Tris-SDS-β-ME sample loading buffer (0.25 M Tris-HCl pH6.8, 2%SDS, 10% β-mercaptoethanol, 30% glycerol, 0.01% Bromophenol Blue) (OwlSeparation, Portsmouth, N.H.) and heated at 100° C. for 5 minutes.Approximately 10 μg of total protein (20 L of heated solution) of samplewas loaded in each of ten lanes on a 12 lane, 10 cm×10 cm×1 mm, 10-20%gradient acrylamide/bis-acrylamide gel (Zaxis, Hudson, Ohio). Molecularweight markers (Novex, San Diego, Calif.) were loaded in the twooutermost lanes of each side of the gel. Electrophoresis was carried outon an Owl Separations Mini-Gel rig at a constant amperage of 50 mA for 1hour in Bio-Rad Tris-Glycine-SDS running buffer. The gel was then rinsedwith deionized water and transferred to Millipore Immobilon-P PVDF(polyvinylidene fluoride) using a semi-dry blotting system supplied byOwl Separations at constant amperage of 150 mA for 1 hour. The resultingblot was stained with Amido Black (10% acetic acid, 0.1% amido black indeionized water) and destained in 10% acetic acid. The protein band wasthen excised from all ten lanes using a methanol cleaned scalpel ormini-Exacto knife and placed in the reaction cartridge of the AppliedBiosystems 477A Protein Sequencer (Foster City, Calif.). The N-terminalSequencer was then run under optimal blot conditions for 12 or morecycles (1 cycle Blank, 1 cycle Standard, and 10 or more cycles fordesired residue identification). PTH-amino acid detection was done onthe Applied Biosystems 120A PTH Analyzer. The cycles were collected bothon an analog chart recorder and digitally via the instrument software.N- terminal Amino acid assignment was perfomed by comparison of theanalog and digital data to a standard set of PTH-amino acids and theirrespective retention times on the analyzer (cysteine residues aredestroyed during conversion and are not detected).

[0173] Subcloning and Expression of the Recombinant 20 kDa SurfaceAssociated Proteins

[0174] N-terminal sequence was compared against the NCBI non redundantdatabase located at www.ncbi.nlm.org using the BLAST algorithimdeveloped by Altschul (Altschul, SF, et al., J. Mol-Biol., 1990,215:403). This showed that the N-terminal sequence had identity to aopen reading frame (ORF) in NCBI database. This ORF had been previouslysequenced and was listed as an unidentified ORF (Pikis, A. et al., J.Infect. Dis., 1998, 178:700). Subsequent BLAST analysis of the unknownORF against the public release of the S. pneumoniae genome (serotype 4),made available by The Institute for Genomic Research (TIGR,www.tigr.org), showed the ORF to be present in the genome, butunidentified as well. DNA analyses of the unknown ORF in the S.pneumoniae genomic sequence and primer designs were performed using theDNASTAR (Madison, Wis.) Lasergene DNA and protein analysis software.

[0175] Primers flanking the ORF were designed (SED ID NOs: 1 and 2) andsubsequently synthesized using the ABI 380A DNA synthesizer. Tofacilitate subcloning the PCR product into the pET28a expression vector,restriction sites were designed into the PCR primers. An Nco1 site wasincluded in the 5′ primer, which allowed both for the ligation into theNco1 site of the expression vector and also included an ATG start codon.To maintain the correct reading frame, two extra bases were included inthe 5′ primer, resulting in the addition of a codon for Leucine. A Sal1site was included in the 3′ primer.

[0176] A PCR fragment of the expected size was generated from CP1200,ligated into the pCR2.1 vector, and used to transform OneShot Top 10F′cells (Invitrogen). Ampicillin resistant transformants were screenedscreened by restriction digestion of plasmid DNA prepared by alkalinelysis (Bimboim, H. C. and Duly, J., Nuc. Acid Res., 1978 7:1513). Arecombinant plasmid, containing the 20 kDa gene, was identified. DNAsequence was obtained from the clones using the Applied Biosystems PrismDye Terminator cycle-sequencing core kit based on the Prism protocolsupplied by the vendor. Approximately 1 ug of template DNA and 100 ng ofprimer were used for each cycling reaction. The reactions were cycled onthe GeneAmp PCR Systems 2400 unit, purified using the Prism method, andanalyzed on an ABI 373A DNA sequencer (Applied Biosystems).

[0177] The insert containing the r20 kDa gene was excised by restrictiondigestion with Nco1 and Sal1, and separated on a 1.5% Agarose gel. TheDNA fragment was cut from the gel and purified away from the agarose bya Bio 101Spin kit (Vista, Calif.). The insert was ligated with plasmidvector DNA(pET28a) also digested with Nco1 and Sal1, and wassubsequently transformed into Top10F′ cells (Invitrogen). The kanamycinresistant transformants were screened by restriction digestion ofplasmid DNA prepared by alkaline lysis (Birnboim, H. C. and Duly J.,Nuc. Acid Res., 1978 7:1513). A recombinant plasmid was subsequentlytransformed into BL21 cells (Novagen) to create pLP533 and grown in SOBmedia supplemented with 30 ug/ml kanamycin. Cells were grown to anO.D.₆₀₀ of 0.6, and were subsequently induced with 0.4 mM IPTG(Boehringer Mannheim, Indianapolis, Ind.) for 2-4 hours. Whole celllysates were prepared and electrophoresed on a 15% SDS-PAGE gel(Laemmli, U. K., Nature, 1970,227:680) to confirm expression of thedesired recombinant product.

[0178] Purification of the Recombinant 20 kDa Surface AssociatedProtein.

[0179] A 250 mL flask containing 50 mL of SOB medium, supplemented with30 μg/ML kanamycin (Sigma, St. Louis, Mo.), was inoculated with ascraping from a frozen culture of E. coli pLP533. The culture wasincubated at 37° C. with shaking at 200 rpm for approximately 16 hours.Subsequently, two 1 liter flasks containing SOB plus 30 ug/ml kanamycinwere inoculated with 20 mL of the overnight culture and incubated at 37°C. with shaking at 200 rpm. When the culture reached an optical densityof OD₆₀₀0.7-0.8, IPTG (Gold Biotechnology, St. Louis, Mo.) was added to0.8 mM. The culture was incubated at the same temperature with shakingfor an additional three hours. The cells were then harvested bycentrifugation for 15 min. at 7300× g. The cell pellets were frozen at−20° C. and were then thawed and resuspended in 300 mL of 10 mM sodiumphosphate pH 6.0 (J. T. Baker, Phillipsburg, Pa. ). The cell suspensionwas then passed through a microfluidizer (Microfluidics Corporation,Newton, Mass.) to lyse the cells. The lysate was centrifuged for 15 min.at 16,000× g and the resulting supernatant was then centrifuged for 45min. at 200,000× g. Supernatants and pellets at each step were assayedby SDS-PAGE. The supematant was diluted to 500 mL in 10 mM sodiumphosphate pH 6.0. The solution was then diafiltered with a 100,000 MWcutoff membrane ( Millipore, Bedford, Mass.) against 1 L of the samebuffer and concentrated 2.5 fold. The protein, in the retentate, wasloaded onto a 70 mL ceramic hydroxyapatite column (Bio-Rad LaboratoriesHercules, Calif.) in 10 mM sodium phosphate pH 6.0. The column was thenwashed with 10 column volumes (CV) of the loading buffer. Contaminatingproteins were removed by washing the column with 10 CV of 108 mM sodiumphosphate pH 6.0. The protein was eluted from the column with a lineargradient over 10 CV from 108 mM to 500 mM sodium phosphate pH 6.0. Thepeak fractions were run on a 10% -20% SDS-PAGE gel (Zaxis, Hudson,Ohio). The fractions containing the protein were pooled and stored at−20° C. The protein was analyzed for homogeneity by SDS-PAGE, and theconcentration of protein during purification was determined by themethod of Lowry (Lowry, O. H., et al, S. Biol. Chem., 1951, 198:265).Protein concentration prior to immunization was determined using a BCAkit obtained from Pierce Chemicals (Northbrook, Ill.) and was usedaccording to the manufacturers directions. BSA was used as proteinstandard.

[0180] Polyclonal Antisera for Western Blot Analysis.

[0181] Recombinant protein was used to generate polyclonal antisera inmice. Briefly, 10 μg of r20 kDa protein was adjuvanted for each dose asan emulsion with Incomplete Freund's Adjuvant (IFA) (1:1v/v) andinjected subcutaneously into 6-8 week old Swiss Webster mice. The micewere bled and vaccinated at wk 0, boosted at wk4, then exsanguinated atwk 6. Ten mice were vaccinated with the r20 kDa protein adjuvanted withIFA. The sera were pooled and used for further analysis.

[0182] SDS-PAGE and Western Blotting.

[0183] Whole cell lysates were prepared by centrifuging equivalentnumbers of pneumococcal cells, based on the OD₆₀₀, in a microcentrifugefor 30 sec. Pneumococcal cell pellets were resuspended in an appropriatevolume of loading buffer. Where indicated, samples were boiled for 5 minand separated on a 10% SDS-PAGE gel using the method of Laemmli(Laemmli, Nature, 1970; 227:680). The samples were transferred tonitrocellulose (BioRad, Hercules, Calif.) using a Biorad Mini Transblotcell (Biorad) and the blots were blocked at room temp for 30 minutes in5% nonfat milk-PBS (BLOTTO). Pooled mouse antisera were used at a 1:1000dilution in BLOTTO for 60 minutes, followed by 25 minute washes inPBS-0.2% Tween80. Goat anti-mouse IgG+M conjugated to alkalinephosphatase (Biosource International, Camarillo, Calif.) was used todetect bound antibodies at a 1:1000 dilution in BLOTTO. The blots werewashed as previously described and detected with NBT and BCIP fromBioRad according to the manufacturer's directions.

[0184] Intranasal Immunization of Mice Prior to Challenge.

[0185] Six-week old, pathogen-free, Balb/c mice were purchased fromJackson Laboratories (Bar Harbor, Me.) and housed in cages understandard temperature, humidity, and lighting conditions. BALB/C mice, at10 animals per group, were immunized with 5 μg of r20 kDa protein. Onweeks 0, 2, and 4. On each occasion, 5 μg r20 kDa formulated with 0.1 μgof CT-E29H, a genetically modified cholera toxin that is reduced inenzymatic activity and toxicity (Tebbey, P. W., et al., Vaccine, 2000,18:2723), was slowly instilled into the nostril of each mouse in a 10 μlvolume. Mice immunized with Keyhole Limpet Hemocyanin (KLH)-CT-E29H wereused as controls. Serum samples were collected 4 days after the lastimmunization.

[0186] Mouse Intranasal Challenge Model.

[0187] Balb/c mice were challenged on week 4 day 6 with 1×10⁵ CFU's ofserotype 3 streptomycin resistant S. pneumoniae. Pneumococci wereinoculated into 3 ml of Todd-Hewitt broth containing 100 μg/ml ofstreptomycin. The culture was grown at 37° C. until mid-log phase, thendiluted to the desired concentration with Todd-Hewitt broth and storedon ice until use. Each mouse was anesthetized with 1.2 mg of ketamineHCl (Fort Dodge Laboratory, Ft. Dodge, Iowa) by i.p. injection. Thebacterial suspension was inoculated to the nostril of anesthetized mice(10 μl per mouse). The actual dose of bacteria administrated wasconfirmed by plate count. Four days after challenge, mice weresacrificed, the noses were removed, and homogenized in 3-ml sterilesaline with a tissue homogenizer (Ultra-Turax T25, Janke & KunkelIka-Labortechnik, Staufen, Germany). The homogenate was 10-fold seriallydiluted in saline and plated on streptomycin containing TSA plates.Fifty μl of blood collected 2 days post-challenge from each mouse wasalso plated on the same kind of plates. Plates were incubated overnightat 37° C. and then colonies were counted.

[0188] ELISA Assay for r20 kDa Protein.

[0189] Antibody titers against r20 kDa protein were determined byenzyme-linked immunosorbent assay (ELISA). ELISAs were performed usingr20 kDa (100 μl per well of a 5 μg/ml stock in PBS, pH7.1) to coatNunc-Immuno™ PolySorp Plates. Plates were coated overnight at 4° C.After blocking with 200 μl of PBS containing 5% nonfat dry milk(blocking buffer) for 1 hour at room temperature, the plates wereincubated with serial dilutions of test sera diluted in blocking bufferfor 1.5 hours at room temperature. The plates were then washed fivetimes with PBS containing 0.1% Tween (PBS-T) and incubated withbiotinylated goat anti-mouse IgG or IgA (1:8000 or 1:4000 in PBS;Brookwood Biomedical, Birmingham, Ala.) for 1 hour at room temperature.After five additional washes with PBS-T, the plates were incubated withstreptavidin conjugated horseradish peroxidase (1:10,000 in PBS; ZymedLaboratory Inc., San Francisco, Calif.) for 1 hour at room temperature.The plates were then washed five times with PBS-T, incubated 20 minuteswith 100 μl of ABTS substrate (KPL, Gaithersburg, Md.), followed byaddition of 100 μl stopping solution (1% SDS). Absorbance values wereread at 405 nm using a VERSAmax microplate reader (Molecular DevicesCorp., Sunnyvale, Calif.). The end point titers of test sera were thereciprocal of the highest mean dilution that resulted in an OD₄₀₅reading of 0.1. The mean background titers of test sera were quantifiedby absorbance values read at 405 nm on the wells that had all reagentsexcept sera.

[0190] Statistical Methods. Comparison of nasal colonization amonggroups was performed using the Tukey-Kramer test (Ludbrook, J., Clin ExpPharmacol Physiol., 1998, 25:1032). Results were considered significantat p<0.05.

[0191] Sequence Heterogeneity of PPP1.

[0192] To examine sequence heterogeneity for the PPP1 protein, thenucleotide sequence for the gene was compared among 10 differentserotypes. Genomic DNA was prepared from overnight cultures of eachserotype of S. pneumoniae. Cells were harvested by centrifugation at1000×g for 15 minutes at 4° C. and resuspended in 2 ml TE buffer. Cellswere lysed by the addition of SDS to 0.3% and Proteinase K (Sigma) to 10μg/ml. The cells were incubated overnight at 55° C. Proteins wereextracted from the cleared lysate by the addition of an equal volume ofphenol/chloroform/ isoamyl alcohol (made by combining a 24:1 mixture ofchloroform/isoamyl alcohol with an equal volume of water saturatedphenol). The phases were separated by centrifugation at 7500 ×g for 10minutes at room temperature, then the aqueous phase was removed to a newtube. The process was repeated, then the DNA was precipitated from theaqueous phase by the addition of 10.4M NH₄Ac to 20%, and 2.5 volume ofethanol. The genomic DNA was spooled out using a glass rod andresuspended in 200 μl TE buffer. The gene for PPP1 was sequenced fromthe genomic DNA of serotypes 1,3,4,5,6,7,9,14,18,23F, and CP1200, usingthe Applied Biosystems Prism Dye Terminator cycle-sequencing core kitbased on the Prism protocol supplied by the vendor. Approximately 1 μgtemplate DNA and 100 ng of primers were used for each cycling reaction.The reactions were cycled on the Gene Amp PCR Systems 2400 unit,purified using the Prism method, and analyzed on an ABI 373A DNAsequencer (Applied Biosystems). The nucleotide sequences and theirpredicted amino acid sequences were aligned in the Megalign applicationof the DNA Lasergene package from DNAstar, using the Clustal Walgorithm.

[0193] Evaluation of PPP1 Message Expressed In Vivo.

[0194] Preparation of RNA from cells grown in vitro

[0195] Various S. pneumoniae serotypes were grown to log phase (O.D.₅₅₀approx 0.3) in 60 ml THB −0.5%YE at 37° C. with 5% CO₂. The cells wereharvested by centrifugation at 1000×g for 15 minutes at 4° C. Thesupernatant was aspirated and the cells were resuspended in 1 ml RNAselater (Ambion, Calif.) and stored for >1 hr at 4° C. The cells were thencentrifuged in a microfuge for 5 minutes at 8000×g. The supernatant wasaspirated and the cells were resuspended in 100 μl 10%Deoxycholate(DOC). 1100 μl of RNAZOL B (Tel-Test, Inc) was then added and thesuspension mixed briefly by inversion. 120 μl of CHCl₃ were then added,the sample mixed by inversion and then centrifuged in a microfuge atfull speed for 10 minutes at 4° C . The aqueous layer was removed andthe RNA was precipitated by addition of an equal volume of 2-propanol.The RNA was incubated at 4° C. for >1 hr and then centrifuged in amicrofuge at full speed for 10 minutes at room temperature. Thesupernatant was aspirated and the RNA was washed with 75% ethanol andrecentrifuged for 5 minutes. The supernatant was aspirated and the RNAwas resuspended in 50-100 μl nuclease free water. DNA was removed fromthe RNA by treating the sample with RNAse free DNAase (DNA FREE, Ambion)for 20 minutes at 37 degrees, followed by inactivation of the enzyme byaddition of the DNA FREE chleator. The purity and yield of the RNA wasassessed by measuring the absorbance at 260 and 280 nm.

[0196] Preparation of RNA from Cells Grown In Vivo

[0197] Log phase S. pneumoniae cells were prepared as described aboveand resuspended to 106 cfu/ml in RPMI media (Celltech) supplemented with0.4% glucose. 1 ml of the cell suspension was sealed in a PVDF dialysismembrane with a 80,000 MW cutoff (SprectraPor). Two such bags wereimplanted intraperitoneally in 400 g Sprague Dawley rats. The bagsremained in the rats for 22 hours, after which the rats were terminatedand the bags were harvested. RNA was prepared from the intraperitoneallygrown cells as described above.

[0198] RT-PCR to Examine the Message for PPP1 In Vivo

[0199] Message for the PPP1 gene was amplified out from both RNAprepared from in vitro and in vivo grown cells using RT-PCR. A reversePCR primer corresponding to the 3′ end of the gene was used to generateds cDNA in the following reaction. 1 μg RNA was incubated with 0.25 μMof the reverse primer: GGG GTC GAC TAA ACC AGG TGC TTG TCC AAG TTC (SEQID NO:8) for 3 minutes at 75° C., then cooled to 44° C. The message wasreverse transcribed using the RETROscript (Ambion) kit according to themanufacturer's directions. ReddyMix (ABgene) was used according to themanufacturer's directions to amplify the PPP1 message from 2-5 μl of thesample, using 0.25 μM of the above reverse primer and the forwardprimer: GGG GCC ATG GCT GTA GAA TTG AAA AAA GAA (SEQ ID NO:9). 10 μl ofthe amplified product was electrophoresed on a 2% Agarose gel.

Results

[0200] Identification of the 20 kDa Surface Associated Protein

[0201] A PBS wash and ion exchange chromatography was used to identifyan 20 kDa surface associated component of S. pneumoniae (FIG. 1). Lane2-9 in FIG. 1 represents fraction #8-16 from a DEAE column. There isclearly a major protein band between 15 and 20 kDa. The low molecularweight band was resolved on a preparative SDS-PAGE gel and transferredto a PVDF membrane. The PVDF membrane has a high binding capacity, whichincreases sample recovery and sequencing performance, allowing efficientdetermination of the amino terminal residues. The amino terminalsequence (SEQ ID NO: 3) of this protein allowed the identification of acorresponding open reading frame in the S. pneumoniae genome (SEQ IDNOS: 4 and 5). This ORF showed similarity to similar to non-hemeiron-containing ferritin proteins in other organisms, which may indicatesimilar function in S. pneumoniae (Pikis, A., et al., J. InfectiousDiseases, 1998, 178:700). However, the exact function and cellularlocation of the proteins in S. pneumoniae is unknown. Subcloning andexpression of this ORF provided recombinant material of the expectedsize (FIG. 2).

[0202] Purification of the Recombinant 20 kDa Surface AssociatedProtein.

[0203] Purification was aided by the solubility of the recombinantprotein. Significant purification away from cellular membranes wasachieved by sequential centrifugations. In addition, the characteristicoligomer formation was successfully utilized to remove the remaining lowmolecular weight contaminating proteins by diafiltration. The predictedcharge of the protein at neutral pH allowed the protein to be purifiedto greater than 90% homogeneity on a Hydroxyapatite column, as seen inFIG. 5.

[0204] Reactivity of Anti-r20 kDa Surface Associated Protein Sera.Polyclonal antisera to recombinant

[0205] 20 kDa surface associated protein were generated in Swiss Webstermice to evaluate antigenic conservation of the protein among strains.Antisera to the r20 kDa protein reacted with proteins of approximately20, 40, and 80 kDa in unheated whole cell lysates of native species(FIG. 3), while the major reactive species seen in heated samples is atapproximately 20 kDa (not shown). These results suggest that thisprotein is part of a complex of 4 subunits or more.

[0206] Intranasal Challenge.

[0207] To determine whether i.n. immunization with r20 kDa surfaceassociated protein can induce serum immune responses, Balb/c mice wereadministered 5 μg r20 kDa 3 times at biweekly intervals using CT-E29H(0.1 μg/dose) as a mucosal adjuvant. Immune sera collected 4 days afterthe last booster immunization were tested in the antigen-specific ELISAassays. At 4 days after the last booster immunization, strong,antigen-specific IgG and IgA antibody responses were generated in miceimmunized with r20 kDa- E29H (Table 6). When compared to the unrelatedprotein KLH, immunization with r20 kDa surface associated protein wasable to significantly reduce colonization of type 3 S. pneumoniae thenasopharynx of BALB/C mice. (FIG. 4) The results are comparable to theability of the type 3 conjugate to reduce colonization of the homologousserotype (Henrikson, J, et al. Alcohol Clin Exp Res, 1997, 21:1630).Antigen specific ELISA titers for r20kDa surface associated protein fromS. pneumoniae. Sera wk4d5 Sera wk4d5 Group IgG IgA 5 μg r20kDa + 0.1 ugCT-E29H 79,726 1563 5 μg Type-3-Conjugate + 0.1 ug CT-E29H <50 <50 5 μgKLH + 0.1 ug CT-E29H <50 <50

[0208] Sequence Alignment of the PPP1 protein from 10 serotypes.

[0209] As shown in FIG. 6, the sequence of PPP1 is largely conservedamong serotypes. As can be seen, serotype 9 is the most divergentserotype. The PP1 isolated from this serotype showed 15 amino aciddifferences from the majority. The remaining serotypes showed less than5 amino acid differences. An overall consensus sequence of PPP1 is shownin FIG. 6 (SEQ ID NO:20).

[0210] RNA Amplification.

[0211] A discrete band of the expected size is seen in both the in vitroand in vivo samples (FIG. 7). The size of the product was estimated tobe full length by comparison to Hae III restriction fragments of LambdaDNA.

[0212] The patents, applications, test methods, and publicationsmentioned herein are hereby incorporated by reference in theirentireties.

[0213] Many variations of the present invention will suggest themselvesto those skilled in the art in light of the above detailed description.All such obvious variations are within the full intended scope of theappended claims.

1 20 1 30 DNA Artificial Sequence 5′ primer 1 ggggccatgg tctttccagtttggtcaaaa 30 2 36 DNA Artificial Sequence 3′ primer 2 ggggtcgacttataaaccag gtgcttgtcc aagttc 36 3 20 PRT Streptococcus pneumoniae 3 ValGlu Leu Lys Lys Glu Ala Val Lys Asp Val Thr Ser Leu Thr Lys 1 5 10 15Ala Ala Pro Val 20 4 537 DNA Streptococcus pneumoniae 4 atgaatgaggtaaagaaaat ggtagaattg aaaaaagaag cagtaaaaga cgtaacatca 60 ttgacaaaagcagcgccagt agcattggca aaaacaaagg aagtcttgaa ccaagctgtt 120 gctgatttgtatgtagctca cgttgctttg caccaagtgc actggtatat gcatggtcgt 180 ggtttccttgtatggcatcc aaaaatggat gagtacatgg aagctcttga cggtcaattg 240 gatgaaatcagtgaacgctt gattacactc ggtggaagcc cattctctac attgacagag 300 ttccttcaaaatagtgaaat cgaagaagaa gctggtgaat accgtaatgt tgaagaaagc 360 ttggaacgtgttcttgttat ctaccgttac ttgtcagaac ttttccaaaa aggtttggat 420 gtcactgatgaagaaggtga cgatgtgaca aacggtatct ttgcaggcgc taaaactgaa 480 acagataaaacaatttggat gcttgcagcc gaacttggac aagcacctgg tttgtaa 537 5 178 PRTStreptococcus pneumoniae 5 Met Asn Glu Val Lys Lys Met Val Glu Leu LysLys Glu Ala Val Lys 1 5 10 15 Asp Val Thr Ser Leu Thr Lys Ala Ala ProVal Ala Leu Ala Lys Thr 20 25 30 Lys Glu Val Leu Asn Gln Ala Val Ala AspLeu Tyr Val Ala His Val 35 40 45 Ala Leu His Gln Val His Trp Tyr Met HisGly Arg Gly Phe Leu Val 50 55 60 Trp His Pro Lys Met Asp Glu Tyr Met GluAla Leu Asp Gly Gln Leu 65 70 75 80 Asp Glu Ile Ser Glu Arg Leu Ile ThrLeu Gly Gly Ser Pro Phe Ser 85 90 95 Thr Leu Thr Glu Phe Leu Gln Asn SerGlu Ile Glu Glu Glu Ala Gly 100 105 110 Glu Tyr Arg Asn Val Glu Glu SerLeu Glu Arg Val Leu Val Ile Tyr 115 120 125 Arg Tyr Leu Ser Glu Leu PheGln Lys Gly Leu Asp Val Thr Asp Glu 130 135 140 Glu Gly Asp Asp Val ThrAsn Gly Ile Phe Ala Gly Ala Lys Thr Glu 145 150 155 160 Thr Asp Lys ThrIle Trp Met Leu Ala Ala Glu Leu Gly Gln Ala Pro 165 170 175 Gly Leu 6 42PRT Homo sapiens 6 Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val HisHis Gln Lys 1 5 10 15 Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn LysGly Ala Ile Ile 20 25 30 Gly Leu Met Val Gly Gly Val Val Ile Ala 35 40 728 PRT Homo sapiens 7 Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu ValHis His Gln Lys 1 5 10 15 Leu Val Phe Phe Ala Glu Asp Val Gly Ser AsnLys 20 25 8 33 DNA Artificial Sequence Reverse primer 8 ggggtcgactaaaccaggtg cttgtccaag ttc 33 9 30 DNA Artificial Sequence Forward primer9 ggggccatgg ctgtagaatt gaaaaaagaa 30 10 176 PRT Streptococcuspneumoniae 10 Met Ala Val Glu Leu Lys Lys Glu Ala Val Lys Asp Val ThrSer Leu 1 5 10 15 Thr Lys Ala Ala Pro Val Ala Leu Ala Lys Thr Lys GluVal Leu Asn 20 25 30 Gln Ala Val Ala Asp Leu Tyr Val Ala His Val Ala LeuHis Gln Val 35 40 45 His Trp Tyr Met His Gly Arg Gly Phe Leu Val Trp HisPro Lys Met 50 55 60 Asp Glu Tyr Met Glu Ala Leu Asp Gly Gln Leu Asp GluIle Ser Glu 65 70 75 80 Arg Leu Ile Thr Leu Gly Gly Ser Pro Phe Ser ThrLeu Thr Glu Phe 85 90 95 Leu Gln Asn Ser Glu Ile Glu Glu Glu Ala Gly GluTyr Arg Asn Val 100 105 110 Glu Glu Ser Leu Glu Arg Val Leu Val Ile TyrArg Tyr Leu Ser Glu 115 120 125 Leu Phe Gln Lys Gly Leu Asp Val Thr AspGlu Glu Gly Asp Asp Val 130 135 140 Thr Asn Gly Ile Phe Ala Gly Ala LysThr Glu Thr Asp Lys Thr Ile 145 150 155 160 Trp Met Leu Ala Ala Glu LeuGly Gln Ala Pro Gly Leu Val Asp Pro 165 170 175 11 176 PRT Streptococcuspneumoniae 11 Met Ala Val Glu Leu Lys Lys Glu Ala Val Lys Asp Val ThrSer Leu 1 5 10 15 Thr Lys Ala Ala Pro Val Ala Leu Ala Lys Thr Lys GluVal Leu Asn 20 25 30 Gln Ala Val Ala Asp Leu Tyr Val Ala His Val Ala LeuHis Gln Val 35 40 45 His Trp Tyr Met His Gly Arg Gly Phe Leu Val Trp HisPro Lys Met 50 55 60 Asp Glu Tyr Met Glu Ala Leu Asp Gly Gln Leu Asp GluIle Ser Glu 65 70 75 80 Arg Leu Ile Thr Leu Gly Gly Ser Pro Phe Ser ThrLeu Thr Glu Phe 85 90 95 Leu Gln Asn Ser Glu Ile Glu Glu Glu Ala Gly GluTyr Arg Asn Val 100 105 110 Glu Glu Ser Leu Glu Arg Val Leu Val Ile TyrArg Tyr Leu Ser Glu 115 120 125 Leu Phe Gln Lys Gly Leu Asp Val Thr AspGlu Glu Gly Asp Asp Val 130 135 140 Thr Asn Gly Ile Phe Ala Gly Ala LysThr Glu Thr Asp Lys Thr Ile 145 150 155 160 Trp Met Leu Ala Ala Glu LeuGly Gln Ala Pro Gly Leu Val Asp Pro 165 170 175 12 176 PRT Streptococcuspneumoniae 12 Met Ala Val Glu Leu Lys Lys Glu Ala Val Lys Asp Val ThrSer Leu 1 5 10 15 Thr Lys Ala Ala Pro Val Ala Leu Ala Lys Thr Lys GluVal Leu Asn 20 25 30 Gln Ala Val Ala Asp Leu Tyr Val Ala His Val Ala LeuHis Gln Val 35 40 45 His Trp Tyr Met His Gly Arg Gly Phe Leu Val Trp HisPro Lys Met 50 55 60 Asp Glu Tyr Met Glu Ala Leu Asp Gly Gln Leu Asp GluIle Ser Glu 65 70 75 80 Arg Leu Ile Thr Leu Gly Gly Ser Pro Phe Ser ThrLeu Thr Glu Phe 85 90 95 Leu Gln Asn Ser Glu Ile Glu Glu Glu Ala Gly GluTyr Arg Asn Val 100 105 110 Glu Glu Ser Leu Glu Arg Val Leu Val Ile TyrArg Tyr Leu Ser Glu 115 120 125 Leu Phe Gln Lys Gly Leu Asp Val Thr AspGlu Glu Gly Asp Asp Val 130 135 140 Thr Asn Gly Ile Phe Glu Gly Ala LysThr Glu Thr Asp Lys Thr Ile 145 150 155 160 Trp Met Leu Ala Ala Glu LeuGly Gln Ala Pro Gly Leu Val Asp Pro 165 170 175 13 175 PRT Streptococcuspneumoniae 13 Ala Val Glu Leu Lys Lys Glu Ala Val Lys Asp Val Thr SerLeu Thr 1 5 10 15 Lys Ala Ala Pro Val Ala Leu Ala Lys Thr Lys Glu ValLeu Asn Gln 20 25 30 Ala Val Ala Asp Leu His Val Ala His Val Ala Leu HisGln Val His 35 40 45 Trp Tyr Met His Gly Arg Gly Phe Leu Val Trp His ProLys Met Asp 50 55 60 Glu Tyr Met Glu Ala Leu Asp Gly Gln Leu Asp Glu ThrSer Glu Arg 65 70 75 80 Leu Ile Thr Leu Gly Gly Ser Pro Phe Ser Thr LeuThr Glu Phe Leu 85 90 95 Gln Asn Ser Glu Ile Glu Glu Glu Ala Gly Glu TyrArg Asn Val Glu 100 105 110 Glu Ser Leu Glu Arg Val Leu Val Ile Tyr ArgTyr Leu Ser Glu Leu 115 120 125 Phe Gln Lys Asp Leu Asp Val Thr Asp GluGlu Gly Asp Asp Val Thr 130 135 140 Asn Gly Ile Phe Ala Gly Ala Lys ThrGlu Thr Asp Lys Thr Ile Trp 145 150 155 160 Met Leu Ala Ala Glu Leu GlyGln Ala Pro Gly Leu Val Asp Pro 165 170 175 14 176 PRT Streptococcuspneumoniae 14 Met Ala Val Glu Leu Lys Lys Glu Ala Val Lys Asp Val ThrSer Leu 1 5 10 15 Thr Lys Ala Ala Pro Val Ala Leu Ala Lys Thr Lys GluVal Leu Asn 20 25 30 Gln Ala Val Ala Asp Leu Tyr Val Ala His Val Ala LeuHis Gln Val 35 40 45 His Trp Tyr Met His Gly Arg Gly Phe Leu Val Trp HisPro Lys Met 50 55 60 Asp Glu Tyr Met Glu Ala Leu Asp Gly Gln Leu Asp GluIle Ser Glu 65 70 75 80 Arg Leu Ile Thr Leu Gly Gly Ser Pro Phe Ser ThrLeu Thr Glu Phe 85 90 95 Leu Gln Asn Ser Glu Ile Glu Glu Glu Ala Gly GluTyr Arg Asn Val 100 105 110 Glu Glu Ser Leu Glu Arg Val Leu Val Ile TyrArg Tyr Leu Ser Glu 115 120 125 Leu Phe Gln Lys Gly Leu Asp Val Thr AspGlu Glu Gly Asp Asp Val 130 135 140 Thr Asn Asp Ile Phe Val Gly Ala LysThr Glu Thr Asp Lys Thr Ile 145 150 155 160 Trp Met Leu Ala Ala Glu LeuGly Gln Ala Pro Gly Leu Val Asp Pro 165 170 175 15 176 PRT Streptococcuspneumoniae 15 Met Ala Val Glu Leu Lys Lys Glu Ala Val Lys Asp Val ThrSer Leu 1 5 10 15 Thr Lys Ala Ala Pro Val Ala Leu Ala Lys Thr Lys GluVal Leu Asn 20 25 30 Gln Ala Val Ala Asp Leu Tyr Val Ala His Val Ala LeuHis Gln Val 35 40 45 His Trp Tyr Met His Gly Arg Gly Phe Leu Val Trp HisPro Lys Met 50 55 60 Asp Glu Tyr Met Glu Ala Leu Asp Gly Gln Leu Asp GluIle Ser Glu 65 70 75 80 Arg Leu Ile Thr Leu Gly Gly Ser Pro Phe Ser ThrLeu Thr Glu Phe 85 90 95 Leu Gln Asn Ser Glu Ile Glu Glu Glu Ala Gly GluTyr Arg Asn Val 100 105 110 Glu Glu Ser Leu Glu Arg Val Leu Val Ile TyrArg Tyr Leu Ser Glu 115 120 125 Leu Phe Gln Lys Gly Leu Asp Val Thr AspGlu Glu Gly Asp Asp Val 130 135 140 Thr Asn Gly Ile Phe Ala Gly Ala LysThr Glu Thr Asp Lys Thr Ile 145 150 155 160 Trp Met Leu Ala Ala Glu LeuGly Gln Ala Pro Gly Leu Val Asp Pro 165 170 175 16 176 PRT Streptococcuspneumoniae 16 Met Ala Val Glu Leu Lys Lys Glu Ala Val Lys Asp Val ThrSer Leu 1 5 10 15 Thr Lys Ala Ala Pro Val Ala Leu Ala Lys Thr Lys GluVal Leu Asn 20 25 30 Gln Ala Val Ala Asp Leu Tyr Val Ala His Val Ala LeuHis Gln Val 35 40 45 His Trp Tyr Met His Gly Arg Gly Phe Leu Val Trp HisPro Lys Met 50 55 60 Asp Glu Tyr Met Glu Ala Leu Asp Gly Gln Leu Asp GluIle Ser Glu 65 70 75 80 Arg Leu Ile Thr Leu Gly Gly Ser Pro Phe Ser ThrLeu Thr Glu Phe 85 90 95 Leu Gln Asn Ser Glu Ile Glu Glu Glu Ala Gly GluTyr Arg Asn Val 100 105 110 Glu Glu Ser Leu Glu Arg Val Leu Val Ile TyrArg Tyr Leu Ser Glu 115 120 125 Leu Phe Gln Lys Gly Leu Asp Val Thr AspGlu Glu Gly Asp Asp Val 130 135 140 Thr Asn Asp Ile Phe Val Gly Ala LysThr Glu Thr Asp Lys Thr Ile 145 150 155 160 Trp Met Leu Ala Ala Glu LeuGly Gln Ala Pro Gly Leu Val Asp Pro 165 170 175 17 176 PRT Streptococcuspneumoniae 17 Met Ala Val Glu Leu Lys Lys Glu Ala Val Lys Asp Val ThrSer Leu 1 5 10 15 Thr Lys Ala Ala Pro Val Ala Leu Ala Lys Thr Lys GluVal Leu Asn 20 25 30 Gln Ala Val Ala Asp Leu Tyr Val Ala His Val Ala LeuHis Gln Val 35 40 45 His Trp Tyr Met His Gly Arg Gly Phe Leu Val Trp HisPro Lys Met 50 55 60 Asp Glu Tyr Met Glu Ala Leu Asp Gly Gln Leu Asp GluIle Ser Glu 65 70 75 80 Arg Leu Ile Thr Leu Gly Gly Ser Pro Phe Ser ThrLeu Thr Glu Phe 85 90 95 Leu Gln Asn Ser Glu Ile Glu Glu Glu Ala Gly GluTyr Arg Asn Val 100 105 110 Glu Glu Ser Leu Glu Arg Val Leu Val Ile TyrArg Tyr Leu Ser Glu 115 120 125 Leu Phe Gln Lys Gly Leu Asp Val Thr AspGlu Glu Gly Asp Asp Val 130 135 140 Thr Asn Gly Ile Phe Ala Gly Ala LysThr Glu Thr Asp Lys Thr Ile 145 150 155 160 Trp Met Leu Ala Ala Glu LeuGly Gln Ala Pro Gly Leu Val Asp Pro 165 170 175 18 176 PRT Streptococcuspneumoniae 18 Met Ala Val Glu Leu Lys Lys Glu Ala Val Lys Asp Val ThrSer Leu 1 5 10 15 Thr Lys Ala Ala Pro Val Ala Leu Ala Lys Thr Lys GluVal Leu Asn 20 25 30 Gln Ala Val Ala Asp Leu Tyr Val Ala His Val Ala LeuHis Gln Val 35 40 45 His Trp Tyr Met His Gly Arg Gly Phe Leu Val Trp HisPro Lys Met 50 55 60 Asp Glu Tyr Met Glu Ala Leu Asp Gly Gln Leu Asp GluIle Ser Glu 65 70 75 80 Arg Leu Ile Thr Leu Gly Gly Ser Pro Phe Ser ThrLeu Thr Glu Phe 85 90 95 Leu Gln Asn Ser Glu Ile Glu Glu Glu Ala Gly GluTyr Arg Asn Val 100 105 110 Glu Glu Ser Leu Glu Arg Val Leu Val Ile TyrArg Tyr Leu Ser Glu 115 120 125 Leu Phe Gln Lys Gly Leu Asp Val Thr AspGlu Glu Gly Asp Asp Val 130 135 140 Thr Asn Gly Ile Phe Ala Gly Ala LysThr Glu Thr Asp Lys Thr Ile 145 150 155 160 Trp Met Leu Ala Ala Glu LeuGly Gln Ala Pro Gly Leu Val Asp Pro 165 170 175 19 176 PRT Streptococcuspneumoniae 19 Met Ala Val Glu Leu Lys Lys Glu Ala Ala Lys Asp Val AlaArg Leu 1 5 10 15 Thr Lys Ala Ala Pro Val Ala Leu Ala Lys Thr Lys GluVal Leu Asn 20 25 30 Gln Ala Val Ala Asp Leu Tyr Val Ala His Val Ala LeuHis Gln Val 35 40 45 His Trp Tyr Met His Gly Arg Gly Phe Leu Val Trp HisPro Lys Met 50 55 60 Asp Glu Tyr Met Glu Ala Leu Asp Gly His Leu Asp GluIle Ser Glu 65 70 75 80 Arg Leu Ile Thr Leu Gly Gly Ser Pro Phe Ser ThrLeu Thr Glu Phe 85 90 95 Leu Gln Asn Ser Glu Ile Glu Glu Glu Ala Gly GluTyr Arg Asn Val 100 105 110 Glu Glu Ser Leu Glu Arg Val Leu Ala Ile TyrArg Tyr Leu Ile Thr 115 120 125 Leu Phe Gln Lys Ala Leu Asp Val Thr AspGlu Glu Gly Asp Asp Val 130 135 140 Thr Asn Asp Ile Phe Val Gly Ala LysAla Glu Leu Glu Lys Thr Val 145 150 155 160 Trp Met Leu Ala Ala Glu LeuGly Gln Ala Pro Gly Leu Val Asp Pro 165 170 175 20 176 PRT ArtificialSequence Consensus sequence 20 Met Ala Val Glu Leu Lys Lys Glu Ala ValLys Asp Val Thr Ser Leu 1 5 10 15 Thr Lys Ala Ala Pro Val Ala Leu AlaLys Thr Lys Glu Val Leu Asn 20 25 30 Gln Ala Val Ala Asp Leu Tyr Val AlaHis Val Ala Leu His Gln Val 35 40 45 His Trp Tyr Met His Gly Arg Gly PheLeu Val Trp His Pro Lys Met 50 55 60 Asp Glu Tyr Met Glu Ala Leu Asp GlyGln Leu Asp Glu Ile Ser Glu 65 70 75 80 Arg Leu Ile Thr Leu Gly Gly SerPro Phe Ser Thr Leu Thr Glu Phe 85 90 95 Leu Gln Asn Ser Glu Ile Glu GluGlu Ala Gly Glu Tyr Arg Asn Val 100 105 110 Glu Glu Ser Leu Glu Arg ValLeu Val Ile Tyr Arg Tyr Leu Ser Glu 115 120 125 Leu Phe Gln Lys Gly LeuAsp Val Thr Asp Glu Glu Gly Asp Asp Val 130 135 140 Thr Asn Gly Ile PheAla Gly Ala Lys Thr Glu Thr Asp Lys Thr Ile 145 150 155 160 Trp Met LeuAla Ala Glu Leu Gly Gln Ala Pro Gly Leu Val Asp Pro 165 170 175

We claim:
 1. An isolated S. pneumoniae surface associated PneumoProtective Protein (PPP) having a molecular weight of about 20 kiloDaltons (kDa), wherein said molecular weight is determined using a10-20% SDS-PAGE gel, or a fragment thereof; said PPP having the abilityto reduce colonization of pneumococcal bacteria.
 2. A PPP as defined inclaim 1, wherein said PPP is a recombinant protein.
 3. A PPP as definedin claim 2, said PPP having an isoelectric point of about 4.587.
 4. APPP as defined in claim 3, said PPP having a charge of about −14.214 atpH
 7. 5. A PPP as defined in claim 1, said PPP having an amino acidsequence as depicted in SEQ ID NO: 5, or a fragment thereof.
 6. Anucleic acid sequence encoding a PPP as defined in claim 1, wherein saidnucleic acid sequence has a sequence as depicted in SEQ ID NO: 4, or afragment thereof.
 7. A nucleic acid as defined in claim 6 which is acDNA.
 8. An expression vector comprising a nucleic acid sequenceencoding a PPP as defined in claim 1, wherein said sequence isoperatively associated with an expression control sequence.
 9. A vectoras defined in claim 8, wherein said PPP has an isoelectric point ofabout 4.587.
 10. A vector as defined in claim 9, wherein said PPP has acharge of about −14.214 at pH7.
 11. An expression vector comprising anucleic acid sequence encoding a PPP as defined in claim 5, wherein saidsequence is operatively associated with an expression control sequence.12. A vector as defined in claim 11, wherein said nucleic acid sequencehas a sequence as sequence as depicted in SEQ ID NO: 4, or a fragmentthereof.
 13. A host cell transfected with the vector as defined in claim8.
 14. A host cell transfected with the vector as defined in claim 11.15. A method for producing recombinant PPP which method comprisesisolating said PPP produced by the host cells as defined in claim 13,wherein the host cells have been cultured under conditions that providefor expression of said PPP by said vector.
 16. A method for producingrecombinant PPP which method comprises isolating said PPP produced bythe host cells as defined in claim 14, wherein the host cells have beencultured under conditions that provide for expression of said PPP bysaid vector.
 17. A composition comprising a PPP as defined in claim 1and a pharmaceutically acceptable carrier.
 18. A composition comprisinga PPP as defined in claim 5 and a pharmaceutically acceptable carrier.19. A composition comprising a nucleic acid sequence encoding a PPP asdefined in claim 6 and a pharmaceutically acceptable carrier.
 20. Acomposition comprising the expression vector as defined in claim 8 and apharmaceutically acceptable carrier.
 21. A composition comprising theexpression vector as defined in claim 11 and a pharmaceuticallyacceptable carrier.
 22. A composition comprising the host cell asdefined in claim 13 and a pharmaceutically acceptable carrier.
 23. Acomposition comprising the host cell as defined in claim 14 and apharmaceutically acceptable carrier.
 24. An immunogenic compositioncomprising (i) a S. pneumoniae surface associated PPP having a molecularweight of about 20 kilo Daltons (kDa), wherein said molecular weight isdetermined using a 10-20% SDS-PAGE gel, or a fragment thereof; (ii) apharmaceutically acceptable carrier; and (iii) optionally at least oneadjuvant.
 25. A composition as defined in claim 24, said PPP having anisoelectric point of about 4.587.
 26. A composition as defined in claim25, said PPP having a charge of about −14.214 at pH7.
 27. A compositionas defined in claim 24, said PPP having an amino acid sequence asdepicted in SEQ ID NO: 5 or an immunogenic fragment thereof.
 28. Acomposition as defined in claim 27, said PPP encoded by a nucleic acidsequence having a sequence as depicted in SEQ ID NO: 4, or animmunogenic fragment thereof.
 29. A composition as defined in claim 24,wherein said composition elicits protective immunity from a diseasecaused by Streptococcus pneumoniae.
 30. A composition as defined inclaim 29, wherein said disease is selected from the group consisting ofotitis media, rhinosinusitis, bacteremia, meningitis, pneumonia, andlower respiratory tract infection.
 31. A composition as defined in claim29, wherein said PPP comprises an amino acid sequence as depicted in SEQID NO: 5, or an immunogenic fragment thereof.
 32. A composition asdefined in claim 29, wherein said PPP is encoded by a nucleic acidsequence as depicted in SEQ ID NO: 4, or an immunogenic fragmentthereof.
 33. An immunogenic composition comprising (i) at least oneexpression vector encoding a PPP having a molecular weight of about 20kDa, wherein said molecular weight is determined using a 10-20% SDS-PAGEgel; (ii) a pharmaceutically acceptable carrier; and (iii) optionally atleast one adjuvant.
 34. A composition as defined in claim 33, whereinsaid pneumococcal bacteria is Streptococcus pneumoniae.
 35. Acomposition as defined in claim 34, wherein said composition elicitsprotective immunity from a disease caused by Streptococcus pneumoniae.36. A composition as defined in claim 35, wherein said disease isselected from the group consisting of otitis media, rhinosinusitis,bacterenia, meningitis, pneumonia, and lower respiratory tractinfection.
 37. A composition as defined in claim 33, said PPP having anisoelectric point of about 4.582.
 38. A composition as defined in claim37, said PPP having a charge of about 14.214 at pH7.
 39. A compositionas defined in claim 33, wherein said expression vector comprises anucleic acid sequence encoding an amino acid sequence as depicted in SEQID NO: 5, or an immunogenic fragment thereof.
 40. A composition asdefined in claim 33, wherein said expression vector comprises a nucleicacid sequence depicted in SEQ ID NO: 4, or an immunogenic fragmentthereof.
 41. A method of inducing an immune response in a mammal, saidmethod comprising administering to said mammal an amount of acomposition as defined in claim 24 effective to induce an immuneresponse in said mammal.
 42. A method of inducing an immune response ina mammal, said method comprising administering to said mammal an amountof a composition as defined in claim 27 effective to induce an immuneresponse in said mammal.
 43. A method of inducing an immune response ina mammal, said method comprising administering to said mammal an amountof a composition as defined in claim 33 effective to induce an immuneresponse in said mammal.
 44. A method of inducing an immune response ina mammal, said method comprising administering to said mammal an amountof a composition as defined in claim 39 effective to induce an immuneresponse in said mammal.
 45. A method of inducing an immune response ina mammal which is infected with pneumococcal bacteria, said methodcomprising administering to said mammal an amount of a compoundeffective to inhibit binding of an amino acid sequence as depicted inSEQ ID NO: 5 to induce said immune response in said mammal.
 46. A methodfor screening for a compound which induces an immune response in amammal infected with pneumococcal bacteria, said method comprisingcomparing a first amount of binding of an amino acid sequence asdepicted in SEQ ID NO: 5 in the presence of said compound to a secondamount of binding of an amino acid sequence as depicted in SEQ ID NO: 5not in the presence of said compound; whereby a lower first amount ofbinding than said second amount binding indicates that said compound mayinduce said immune response in said mammal.
 47. A method for diagnosingpneumococcal bacterial infection, said method comprising comparing thelevel of PPP as depicted in SEQ ID NO: 5, or fragments thereof, insuspect sample to the level of PPP as depicted in SEQ ID NO: 5, orfragments thereof, in a control sample, whereby a higher level of saidPneumo Protective Protein said suspect sample than the level of saidPneumo Protective Protein in said control sample indicates that saidsuspect sample comprises pneumococcal bacterial infection.
 48. Anantibody which binds to Streptococcus pneumoniae PPP.
 49. An antibody asdefined in claim 48, which selectively recognizes an amino acid sequenceas depicted in SEQ ID NO: 5, or fragments thereof.
 50. An antibody asdefined in claim 48, which is chimeric.
 51. An antibody as defined inclaim 48, which is humanized.
 52. An antibody as defined in claim 48,which is anti-idiotypic.
 53. An antibody as defined in claim 48, whichis conjugated to a pharmaceutically active compound.
 54. An antibody asdefined in claim 48, which is a monoclonal antibody.
 55. An antibody asdefined in claim 54, which is humanized.
 56. An antibody as defined inclaim 54, which is anti-idiotypic.
 57. An antibody as defined in claim54, which is conjugated to a pharmaceutically active compound.
 58. Amethod for inducing an immune response in a mammal, said methodcomprising administering to said mammal an amount of an antibody asdefined in claim 52 effective to induce an immune response in saidmammal.
 59. A method for inducing an immune response in a mammal, saidmethod comprising administering to said mammal an amount of an antibodyas defined in claim 56 effective to induce an immune response in saidmammal.
 60. A method for inducing an immune response in a mammalinfected with pneumococcal bacteria, said method comprisingadministering to said mammal an amount of an antibody as defined inclaim 53 effective to induce an immune response in said mammal.
 61. Amethod for inducing an immune response in a mammal infected withpneumococcal bacteria, said method comprising administering to saidmammal an amount of an antibody as defined in claim 57 effective toinduce an immune response in said mammal.